Cell-permeable (icp)-socs3 recombinant protein and uses thereof

ABSTRACT

The present invention relates to providing improved cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof. Preferably, the iCP-SOCS3 recombinant protein may be used as protein-based anti-hepatocellular carcinoma (HCC) agent by utilizing the platform technology for macromolecule intracellular transduction.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application is a Continuation-in-Part of U.S. application Ser. No. 14/838,260 filed Aug. 27, 2015, which claims the benefit of U.S. Provisional Application No. 62/042,493, filed on Aug. 27, 2014 in the United States Patent and Trademark Office, the disclosures of which are incorporated herein by reference in their entireties.

TECHNICAL FIELD

The present invention relates to providing improved cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof. Preferably, the iCP-SOCS3 recombinant protein may be used as protein-based anti-hepatocellular carcinoma (HCC) agent by utilizing the platform technology for macromolecule intracellular transduction.

BACKGROUND ART

Hepatocellular carcinoma (HCC) is one of the most common cancers with high mobility/mortality rate and the tumor often develops after liver cirrhosis and advanced fibrosis. Cytokines including IL-6 and interferon-gamma (IFN-γ) activate the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway, a vital role promoting the inflammation, fibrosis and carcinogenesis in the liver. STATS, which functions as an oncogene downstream of IL-6/gp130, is hyper-activated in hepatocellular carcinoma contributes to increase cell proliferation and inhibits apoptosis by inducing c-MYC, Cyclin-D1, and Bcl-XL.

Cytokine signaling is strictly regulated by the suppressor of cytokine signaling (SOCS) family proteins induced by different classes of agonists, including cytokines, hormones and infectious agents. Among them, SOCS1 and SOCS3 are relatively specific to STAT1 and STAT3, respectively. SOCS1 inhibits JAK activation through its N-terminal kinase inhibitory region (KIR) by the direct binding to the activation loop of JAKs, while SOCS3 binds to janus kinases (JAKs)-proximal sites on the receptor through its SH2 domain and inhibits JAK activity that blocks recruitment of STAT3. Both promote anti-inflammatory effects due to the suppression of inflammation-inducing cytokine signaling. Furthermore, the SOCS box, another domain in SOCS proteins, interacts with E3 ubiquitin ligases and/or couples the SH2 domain-binding proteins to the ubiquitin-proteasome pathway. Therefore, SOCSs inhibit cytokine signaling by suppressing JAK kinase activity and degrading the activated cytokine receptor complex.

In connection with SOCSs and HCC, the SOCS1 gene has been implicated as an anti-oncogene in the HCC development. Previous studies have reported that aberrant methylation in the CpG island of SOCS1 induces its transcriptional silencing in HCC cell lines, and SOCS1 heterozygous mice are hypersensitive to hepatocellular carcinogenesis. In addition, abnormalities of SOCS3 are also associated with the hepatocellular carcinoma. Hypermethylation of CpG islands in the SOCS3 promoter is correlated with its transcriptional silencing in tumors and in HCC cell lines. SOCS3 overexpression down-regulates active STAT3, induces apoptosis, and suppresses growth in cancer cells. The importance of STAT3 to inflammation-associated hepatocellular carcinogenesis is underlined by the previous study that hepatocyte-specific deletion of SOCS3 in a mouse hepatocellular carcinoma model results in larger and more numerous tumors. This means that SOCS3 plays a major role in the negative regulation of the JAK/STAT pathway in hepatocarcinogenesis and contributes to the suppression of HCC development by protecting the liver cells.

REFERENCES

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DISCLOSURE Technical Problem

To negatively control JAK/STAT signaling, recombinant SOCS3 proteins that contain a cell-penetrating peptide (CPP)-membrane-translocating motif (MTM) from fibroblast growth factor (FGF)-4 has been reported. These recombinant SOCS3 proteins inhibited STAT phosphorylation, inflammatory cytokines production and MHC-II expression in cultured and primary macrophages. In addition, SOCS3 fused to MTM protected mice challenged with a lethal dose of the SEB super-antigen, by suppressing apoptosis and hemorrhagic necrosis in multiple organs. However, the SOCS3 proteins fused to FGF4-derived MTM displayed extremely low solubility, poor yields and relatively low cell- and tissue-permeability. Therefore, the MTM-fused SOCS3 proteins were not suitable for further clinical development as therapeutic agents.

Technical Solution

For MITT, six critical factors (length, bending potential, instability index, aliphatic index, GRAVY, amino acid composition) have been determined through analysis of baseline hydrophobic CPPs. Advanced macromolecule transduction domain (aMTD), newly designed based on these six critical factors, could optimize cell-/tissue-permeability of SOCS3 proteins that have a therapeutic effects and develop them as protein-based drugs. Further, in order to increase solubility and yield of recombinant protein, solubilization domains (SDs) additionally fused to the aMTD-SOCS3 recombinant protein, thereby notably increased the solubility and manufacturing yield of the recombinant protein.

In this application, aMTD/SD-fused iCP-SOCS3 recombinant proteins (iCP-SOCS3), much improved physicochemical characteristics (solubility and yield) and functional activity (cell-/tissue-permeability) compared with the protein fused only to FGF-4-derived MTM. In addition, the newly developed iCP-SOCS3 proteins have now been demonstrated to have therapeutic application in treating hepatocellular carcinoma, exploiting the ability of SOCS3 to suppress JAK/STAT signaling. The present application represents that macromolecule intracellular transduction technology (MITT) enabled by the new hydrophobic CPPs that are aMTD may provide novel protein therapy through SOCS3-intracellular protein replacement against the hepatocellular carcinoma. These findings suggest that restoration of SOCS3 by replenishing the intracellular SOCS3 with iCP-SOCS3 protein creates a new paradigm for anti-cancer therapy, and the intracellular protein replacement therapy with the SOCS3 recombinant protein fused to the combination of aMTD and SD pair may be useful to treat the HCC.

One aspect disclosed in the present application provides an improved Cell-Permeable (iCP)-SOCS3 recombinant protein, which comprises a SOCS3 protein; and at least one advanced macromolecule transduction domain (aMTD)(s) being composed of 9˜13 amino acid sequences and having improved cell and/or tissue permeability, wherein the aMTD(s) is fused to one end or both ends of the SOCS3 protein and has the following features of:

(a) being composed of 3 or more amino acid sequences selected from the group consisting of Ala, Val, Ile, Leu, and Pro;

(b) having proline as amino acid sequences corresponding to any one or more of positions 5 to 8, and 12 of its amino acid sequence; and

(c) having an instability index of 40-60; an aliphatic index of 180-220; and a grand average of hydropathy (GRAVY) of 2.1-2.6, as measured by Protparam.

According to one embodiment, the iCP-SOCS3 recombinant protein further comprises one or more solubilization domain (SD)(s), and the aMTD(s), SOCS3 protein and SD(s) may be randomly fused to one another.

According to another embodiment, the aMTD may form α-Helix structure. According to still another embodiment, the aMTD may be composed of 12 amino acid sequences and represented by the following general formula:

wherein X(s) independently refer to Alanine (A), Valine (V), Leucine (L) or Isoleucine (I); and Proline (P) can be positioned in one of U(s) (either 5′, 6′, 7′ or 8′). The remaining U(s) are independently composed of A, V, L or I, P at the 12′ is Proline.

Another aspect disclosed in the present application provides an iCP-SOCS3 recombinant protein which is represented by any one of the following structural formulae:

A-B-C, A-C-B, B-A-C, B-C-A, C-A-B, C-B-A, A-C-B-C and other possible combinations,

wherein A is an advanced macromolecule transduction domain (aMTD) having improved cell and/or tissue permeability, B is a SOCS3 protein, and C is a solubilization domain (SD); and

the aMTD is composed of 9˜13 amino acid sequences and has the following features of:

(a) being composed of 3 or more amino acids selected from the group consisting of Ala, Val, Ile, Leu, and Pro;

(b) having proline as amino acid sequences corresponding to any one or more of positions 5 to 8, and 12 of its amino acid sequence;

(c) having an instability index of 40-60; an aliphatic index of 180-220; and a grand average of hydropathy (GRAVY) of 2.1-2.6, as measured by Protparam; and

(d) forming α-Helix structure.

According to one embodiment disclosed in the present application, the SOCS3 protein may have an amino acid sequence of SEQ ID NO: 814.

According to another embodiment disclosed in the present application, the SOCS3 protein may be encoded by a polynucleotide sequence of SEQ ID NO: 815.

According to still another embodiment disclosed in the present application, the SOCS3 protein may further include a ligand selectively binding to a receptor of a cell, a tissue, or an organ.

According to still another embodiment disclosed in the present application, the at least one aMTD(s) may have an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 1˜240 and 822.

According to still another embodiment disclosed in the present application, the at least one aMTD(s) may be encoded by a polynucleotide sequence independently selected from the group consisting of SEQ ID NOs: 241˜480 and 823.

According to still another embodiment disclosed in the present application, the one or more SD(s) may have an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 798, 799, 800, 801, 802, 803, and 804.

According to still another embodiment disclosed in the present application, the one or more SD(s) may be encoded by a polynucleotide sequence independently selected from the group consisting of SEQ ID NOs: 805, 806, 807, 808, 809, 810, and 811.

According to still another embodiment disclosed in the present application, the iCP-SOCS3 recombinant protein may have a histidine-tag affinity domain additionally fused to one end thereof.

According to still another embodiment disclosed in the present application, the histidine-tag affinity domain may have an amino acid sequence of SEQ ID NO: 812.

According to still another embodiment disclosed in the present application, the histidine-tag affinity domain may be encoded by a polynucleotide sequence of SEQ ID NO: 813.

According to still another embodiment disclosed in the present application, the fusion may be formed via a peptide bond or a chemical bond.

According to still another embodiment disclosed in the present application, the iCP-SOCS3 recombinant protein may be used for the treating, preventing, or delaying the onset of, hepatocellular carcinoma.

Still another aspect disclosed in the present application provides a polynucleotide sequence encoding the iCP-SOCS3 recombinant protein.

According to one embodiment disclosed in the present application, the polynucleotide sequence may be a polynucleotide sequence represented by SEQ ID NO: 825.

Still another aspect disclosed in the present application provides a recombinant expression vector including the polynucleotide sequence.

Still another aspect disclosed in the present application provides a transformant transformed with the recombinant expression vector.

Still another aspect disclosed in the present application provides a preparing method of the iCP-SOCS3 recombinant protein including preparing the recombinant expression vector; preparing the transformant using the recombinant expression vector; culturing the transformant; and recovering the recombinant protein expressed by the culturing.

Still another aspect disclosed in the present application provides a composition including the iCP-SOCS3 recombinant protein as an active ingredient.

Still another aspect disclosed in the present application provides a pharmaceutical composition for the treating, preventing, or delaying the onset of, hepatocellular carcinoma including the iCP-SOCS3 recombinant protein as an active ingredient; and a pharmaceutically acceptable carrier.

Still another aspect disclosed in the present application provides use of the iCP-SOCS3 recombinant protein as a medicament for the treating, preventing, or delaying the onset of, hepatocellular carcinoma.

Still another aspect disclosed in the present application provides a medicament including the iCP-SOCS3 recombinant protein.

Still another aspect disclosed in the present application provides use of the iCP-SOCS3 recombinant protein in the preparation of a medicament for the treating, preventing, or delaying the onset of, hepatocellular carcinoma.

Still another aspect disclosed in the present application provides a method of treating, preventing, or delaying the onset of, hepatocellular carcinoma in a subject, the method including identifying a subject in need of the treating, preventing, or delaying the onset of, hepatocellular carcinoma; and administering to the subject a therapeutically effective amount of the iCP-SOCS3 recombinant protein.

According to one embodiment disclosed in the present application, the subject may be a mammal.

Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Although a certain method and a material is described herein, it should not be construed as being limited thereto, any similar or equivalent method and material to those may also be used in the practice or testing of the present invention. All publications mentioned herein are incorporated herein by reference in their entirety to disclose and describe the methods and/or materials in connection with which the publications are cited. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

A “peptide,” as used herein, refers to a chain-type polymer formed by amino acid residues which are linked to each other via peptide bonds, and used interchangeably with “polypeptide.” Further, a “polypeptide” includes a peptide and a protein.

Further, the term “peptide” includes amino acid sequences that are conservative variations of those peptides specifically exemplified herein. The term “conservative variation,” as used herein, denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include substitution of one hydrophobic residue, such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine, or methionine for another, or substitution of one polar residue for another, for example, substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like. Neutral hydrophilic amino acids which may be substituted for one another include asparagine, glutamine, serine, and threonine.

The term “conservative variation” also includes use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Such conservative substitutions are within the definition of the classes of the peptides disclosed in the present application.

A person having ordinary skill in the art may make similar substitutions to obtain peptides having higher cell permeability and a broader host range. For example, one aspect disclosed in the present application provides peptides corresponding to amino acid sequences (e.g. SEQ ID NOs: 1 to 240 and 822) provided herein, as well as analogues, homologs, isomers, derivatives, amidated variations, and conservative variations thereof, as long as the cell permeability of the peptide remains.

Minor modifications to primary amino acid sequence disclosed in the present application may result in peptides which have substantially equivalent or enhanced cell permeability, as compared to the specific peptides described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous.

All peptides may be synthesized using L-amino acids, but D forms of all of the peptides may be synthetically produced. In addition, C-terminal derivatives, such as C-terminal methyl esters and C-terminal amidates, may be produced in order to increase the cell permeability of the peptide according to one embodiment disclosed in the present application.

All of the peptides produced by these modifications are included herein, as long as in the case of amidated versions of the peptide, the cell permeability of the original peptide is altered or enhanced such that the amidated peptide is therapeutically useful. It is envisioned that such modifications are useful for altering or enhancing cell permeability of a particular peptide.

Furthermore, deletion of one or more amino acids may also result in a modification to the structure of the resultant molecule without any significant change in its cell permeability. This may lead to the development of a smaller active molecule which may also have utility. For example, amino- or carboxyl-terminal amino acids which may not be required for the cell permeability of a particular peptide may be removed.

The term “gene” refers to an arbitrary nucleic acid sequence or a part thereof having a functional role in protein coding or transcription, or regulation of other gene expression. The gene may be composed of all nucleic acids encoding a functional protein or a part of the nucleic acid encoding or expressing the protein. The nucleic acid sequence may include a gene mutation in exon, intron, initiation or termination region, promoter sequence, other regulatory sequence, or a unique sequence adjacent to the gene.

The term “primer” refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA target polynucleotide and serves as the starting points for the stepwise synthesis of a polynucleotide from mononucleotides by the action of a nucleotidyltransferase as occurs, for example, in a polymerase chain reaction.

The term “coding region” or “coding sequence” refers to a nucleic acid sequence, a complement thereof, or a part thereof which encodes a particular gene product or a fragment thereof for which expression is desired, according to the normal base pairing and codon usage relationships. Coding sequences include exons in genomic DNA or immature primary RNA transcripts, which are joined together by the cellular biochemical machinery to provide a mature mRNA. The anti-sense strand is the complement of the nucleic acid, and the coding sequence may be deduced therefrom.

One aspect disclosed in the present application provides an iCP-SOCS3 recombinant protein, which comprises a SOCS3 protein and at least one advanced macromolecule transduction domain (aMTD)(s) being composed of 9˜13 amino acid sequences, preferably 10˜12 amino acid sequences, and having improved cell and/or tissue permeability, wherein the aMTD is fused to one end or both ends of the SOCS3 protein and has the following features of:

(a) being preferably composed of 3 or more amino acid sequences selected from the group consisting of Ala, Val, Ile, Leu, and Pro;

(b) having proline as amino acid sequences corresponding to any one or more of positions 5 to 8, and 12 of its amino acid sequence, and preferably one or more of positions 5 to 8 and position 12 of its amino acid sequence; and

(c) having an instability index of preferably 40-60 and more preferably 41-58; an aliphatic index of preferably 180-220 and more preferably 185-225; and a grand average of hydropathy (GRAVY) of preferably 2.1-2.6 and more preferably 2.2-2.6 as measured by Protparam (see http://web.expasy.org/protparam/).

These critical factors that facilitate the cell permeable ability of aMTD sequences were analyzed, identified, and determined according to one embodiment disclosed in the present application. These aMTD sequences are artificially assembled based on the critical factors (CFs) determined from in-depth analysis of previously published hydrophobic CPPs.

The aMTD sequences according to one aspect disclosed in the present application are the first artificially developed cell permeable polypeptides capable of mediating the transduction of biologically active macromolecules—including peptides, polypeptides, protein domains, or full-length proteins—through the plasma membrane of cells.

According to one embodiment, the iCP-SOCS3 recombinant protein further comprises one or more solubilization domain (SD)(s), and the aMTD(s), SOCS3 protein and SD(s) may be randomly fused to one another. For example, SD(s) may be further fused to one or more of the SOCS3 protein and the aMTD, preferably to one end or both ends of the SOCS3 protein, and more preferably to the C-terminus of the SOCS3 protein.

According to another embodiment, the aMTD may form α-Helix structure.

According to still another embodiment, the aMTD may be preferably composed of 12 amino acid sequences and represented by the following general formula:

Here, X(s) independently refer to Alanine (A), Valine (V), Leucine (L) or Isoleucine (I); and Proline (P) can be positioned in one of U(s) (either 5′, 6′, 7′ or 8′). The remaining U(s) are independently composed of A, V, L or I, P at the 12′ is Proline.

Still another aspect disclosed in the present application provides an iCP-SOCS3 recombinant protein which is represented by any one of structural formulae A-B-C, A-C-B, B-A-C, B-C-A, C-A-B, C-B-A, A-C-B-C and other possible combinations, preferably by A-B-C or C-B-A:

wherein A is an advanced macromolecule transduction domain (aMTD) having improved cell and/or tissue permeability, and if the iCP-SOCS3 recombinant protein comprises two or more aMTDs, they can be same or different; B is a SOCS3 protein; and C is a solubilization domain (SD), and if the iCP-SOCS3 recombinant protein comprises two or more SDs, they can be same or different; and

the aMTD is composed of 9˜13, preferably 10˜12 amino acid sequences and has the following features of:

(a) being composed of 3 or more amino acid sequences selected from the group consisting of Ala, Val, Ile, Leu, and Pro;

(b) having proline as amino acid sequences corresponding to any one or more of positions 5 to 8, and 12 of its amino acid sequence, and preferably, one or more of positions 5 to 8 and position 12 of its amino acid sequence;

(c) having an instability index of 40-60, preferably 41-58 and more preferably 50-58; an aliphatic index of 180-220. preferably 185-225 and more preferably 195-205; and a grand average of hydropathy (GRAVY) of 2.1-2.6 and preferably 2.2-2.6, as measured by Protparam (see http://web.expasy.org/protparam/); and

(d) preferably forming α-Helix structure.

In one embodiment disclosed in the present application, the SOCS3 protein may have an amino acid sequence of SEQ ID NO: 814.

In another embodiment disclosed in the present application, the SOCS3 protein may be encoded by a polynucleotide sequence of SEQ ID NO: 815.

When the iCP-SOCS3 recombinant protein is intended to be delivered to a particular cell, tissue, or organ, the SOCS3 protein may form a fusion product, together with an extracellular domain of a ligand capable of selectively binding to a receptor which is specifically expressed on the particular cell, tissue, or organ, or monoclonal antibody (mAb) capable of specifically binding to the receptor or the ligand and a modified form thereof.

The binding of the peptide and a biologically active substance may be formed either by indirect linkage by a cloning technique using an expression vector at a nucleotide level or by direct linkage via chemical or physical covalent or non-covalent bond of the peptide and the biologically active substance.

In still another embodiment disclosed in the present application, the SOCS3 protein may preferably further include a ligand selectively binding to a receptor of a cell, a tissue, or an organ.

In one embodiment disclosed in the present application, the at least one aMTD(s) may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 1˜240 and 822, preferably SEQ ID NOs: 2, 16, 22, 32, 40, 43, 63, 65, 77, 84, 85, 86, 110, 131, 142, 143, 177, 228, 229, 233, 237, 239 and 822, more preferably SEQ ID NO: 43.

In still another embodiment disclosed in the present application, the at least one aMTD(s) may be encoded by a polynucleotide sequence independently selected from the group consisting of SEQ ID NOs: 241˜480 and 823, preferably SEQ ID NOs: 242, 256, 262, 272, 280, 283, 303, 305, 317, 324, 325, 326, 350, 371, 382, 383, 417, 468, 469 473, 477, 479 and 823, more preferably SEQ ID NO: 283.

In still another embodiment disclosed in the present application, the one or more SD(s) may have an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 798, 799, 800, 801, 802, 803, and 804. The SD may be preferably SDA of SEQ ID NO: 798, SDB of SEQ ID NO: 799, or SDB′ of SEQ ID NO: 804, and more preferably, SDB of SEQ ID NO: 799 which has superior structural stability, or SDB′ of SEQ ID NO: 804 which has a modified amino acid sequence of SDB to avoid immune responses upon in vivo application. The modification of the amino acid sequence in SDB may be replacement of an amino acid residue, Valine, corresponding to position 28 of the amino acid sequence of SDB (SEQ ID NO: 799) by Leucine.

In still another embodiment disclosed in the present application, the at least one SDs may be encoded by a polynucleotide sequence independently selected from the group consisting of SEQ ID NOs: 805, 806, 807, 808, 809, 810, and 811. The SD may be preferably SDA encoded by a polynucleotide sequence of SEQ ID NO: 805, SDB encoded by a polynucleotide sequence of SEQ ID NO: 806, or SDB′ for deimmunization (or humanization) encoded by a polynucleotide sequence of SEQ ID NO: 811, and more preferably, SDB having superior structural stability, which is encoded by a polynucleotide sequence of SEQ ID NO: 806, or SDB′ having a modified polynucleotide sequence of SDB to avoid immune responses upon in vivo application, which is encoded by a polynucleotide sequence of SEQ ID NO: 811.

In still another embodiment disclosed in the present application, the iCP-SOCS3 recombinant protein may be preferably selected from the group consisting of:

1) a recombinant protein, in which SOCS3 having an amino acid sequence of SEQ ID NO: 814 is fused to the N-terminus or the C-terminus of aMTD having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 240 and 822, 2, 16, 22, 32, 40, 43, 63, 65, 77, 84, 85, 86, 110, 131, 142, 143, 177, 228, 229, 233, 237, 239 and 822, more preferably SEQ ID NO: 43;

2) a recombinant protein, in which SD having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 798 to 804 is further fused to one or more of the N-terminus or the C-terminus of the SOCS3 and aMTD in the recombinant protein of 1); and

3) a recombinant protein, in which a Histidine tag having an amino acid sequence of 812 is further fused to the N-terminus of the recombinant protein of 1) or 2).

According to one embodiment, the iCP-SOCS3 recombinant protein may be composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 825.

The SOCS3 protein may exhibit a physiological phenomenon-related activity or a therapeutic purpose-related activity by intracellular or in-vivo delivery. The recombinant expression vector may include a tag sequence which makes it easy to purify the recombinant protein, for example, consecutive histidine codon, maltose binding protein codon, Myc codon, etc., and further include a fusion partner to enhance solubility of the recombinant protein, etc. Further, for the overall structural and functional stability of the recombinant protein or flexibility of the proteins encoded by respective genes, the recombinant expression vector may further include one or more glycine, proline, and spacer amino acid or polynucleotide sequences including AAY amino acids. Furthermore, the recombinant expression vector may include a sequence specifically digested by an enzyme in order to remove an unnecessary region of the recombinant protein, an expression regulatory sequence, and a marker or reporter gene sequence to verify intracellular delivery, but is not limited thereto.

In still another embodiment disclosed in the present application, the iCP-SOCS3 recombinant protein may preferably have a histidine-tag affinity domain additionally fused to one end thereof.

In still another embodiment disclosed in the present application, the histidine-tag affinity domain may have an amino acid sequence of SEQ ID NO: 812.

In still another embodiment disclosed in the present application, the histidine-tag affinity domain may be encoded by a polynucleotide sequence of SEQ ID NO: 813.

In still another embodiment disclosed in the present application, the fusion may be formed via a peptide bond or a chemical bond.

The chemical bond may be preferably selected from the group consisting of disulfide bonds, diamine bonds, sulfide-amine bonds, carboxyl-amine bonds, ester bonds, and covalent bonds.

In still another embodiment disclosed in the present application, the iCP-SOCS3 recombinant protein may be used for the treating, preventing, or delaying the onset of, hepatocellular carcinoma.

Still another aspect disclosed in the present application provides a polynucleotide sequence encoding the iCP-SOCS3.

According to still another embodiment disclosed in the present application, the polynucleotide sequence may be fused with a histidine-tag affinity domain.

Still another aspect disclosed in the present application provides a recombinant expression vector including the polynucleotide sequence.

Preferably, the vector may be inserted in a host cell and recombined with the host cell genome, or refers to any nucleic acid including a nucleotide sequence competent to replicate spontaneously as an episome. Such a vector may include a linear nucleic acid, a plasmid, a phagemid, a cosmid, an RNA vector, a viral vector, etc.

Preferably, the vector may be genetically engineered to incorporate the nucleic acid sequence encoding the recombinant protein in an orientation either N-terminal and/or C-terminal to a nucleic acid sequence encoding a peptide, a polypeptide, a protein domain, or a full-length protein of interest, and in the correct reading frame so that the recombinant protein consisting of aMTD, SOCS3 protein, and preferably SD may be expressed. Expression vectors may be selected from those readily available for use in prokaryotic or eukaryotic expression systems.

Standard recombinant nucleic acid methods may be used to express a genetically engineered recombinant protein. The nucleic acid sequence encoding the recombinant protein according to one embodiment disclosed in the present application may be cloned into a nucleic acid expression vector, e.g., with appropriate signal and processing sequences and regulatory sequences for transcription and translation, and the protein may be synthesized using automated organic synthetic methods. Synthetic methods of producing proteins are described in, for example, the literature [Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis by Gregg B. Fields (Editor), Sidney P. Colowick, Melvin I. Simon (Editor), Academic Press (1997)].

In order to obtain high level expression of a cloned gene or nucleic acid, for example, a cDNA encoding the recombinant protein according to one embodiment disclosed in the present application, the recombinant protein sequence may be typically subcloned into an expression vector that includes a strong promoter for directing transcription, a transcription/translation terminator, and in the case of a nucleic acid encoding a protein, a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and are described, e.g., in the literatures [Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3d Edition, Cold Spring Harbor Laboratory, N.Y. (2001); and Ausubel, et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. (1989)]. Bacterial expression systems for expression of the recombinant protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22: 229-235 (1983); Mosbach et al., Nature 302: 543-545 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. The eukaryotic expression vector may be preferably an adenoviral vector, an adeno-associated vector, or a retroviral vector.

Generally, the expression vector for expressing the cell permeable recombinant protein according to one embodiment disclosed in the present application in which the cargo protein, i.e. ΔSOCS3 protein, is attached to the N-terminus, C-terminus, or both termini of aMTD may include regulatory sequences including, for example, a promoter, operably attached to a sequence encoding the advanced macromolecule transduction domain. Non-limiting examples of inducible promoters that may be used include steroid-hormone responsive promoters (e.g., ecdysone-responsive, estrogen-responsive, and glutacorticoid-responsive promoters), tetracycline “Tet-On” and “Tet-Off” systems, and metal-responsive promoters.

The polynucleotide sequence according to one embodiment disclosed in the present application may be present in a vector in which the polynucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the polynucleotide sequence by a suitable host cell.

According to one embodiment disclosed in the present application, the polynucleotide sequence may be selected from the following groups:

1) a polynucleotide sequence, in which any one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 241˜480 and 823, preferably SEQ ID NOs: 242, 252, 274, 279, 322, 331, 338, 345, 347, 361, 365, 370, 371, 383, 387, 417, 462, 468, 469, 473, 477, 479 and 823, more preferably SEQ ID NO: 283, is operably linked with a polynucleotide sequence of SEQ ID NO: 815; and

2) a polynucleotide sequence, in which any one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 805 to 811 is further operably linked to the polynucleotide sequence of 1), or further operably linked to between: any one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 241˜480 and 823, preferably SEQ ID NOs: 242, 256, 262, 272, 280, 283, 303, 305, 317, 324, 325, 326, 350, 371, 382, 383, 417, 468, 469, 473, 477, 479 and 823, more preferably SEQ ID NO: 283; and a polynucleotide sequence of SEQ ID NO: 815.

Within an expression vector, the term “operably linked” is intended to mean that the polynucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the polynucleotide sequence. The term “regulatory sequence” is intended to include promoters, enhancers, and other expression control elements. Such operable linkage with the expression vector can be achieved by conventional gene recombination techniques known in the art, while site-directed DNA cleavage and linkage are carried out by using conventional enzymes known in the art.

The expression vectors may contain a signal sequence or a leader sequence for membrane targeting or secretion, as well as regulatory sequences such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, an enhancer and the like. The promoter may be a constitutive or an inducible promoter. Further, the expression vector may include one or more selectable marker genes for selecting the host cell containing the expression vector, and may further include a polynucleotide sequence that enables the vector to replicate in the host cell in question.

The expression vector constructed according to one embodiment disclosed in the present application may be the vector where the polynucleotide encoding the iCP-SOCS3 recombinant protein (where an aMTD is fused to the N-terminus or C-terminus of a SOCS3 protein) is inserted within the multiple cloning sites (MCS), preferably within the Nde1/Sal1 site or BamH1/Sal1 site of a pET-28a(+) (Novagen, Darmstadt, Germany) or pET-26b(+) vector (Novagen, Darmstadt, Germany).

In still another embodiment disclosed in the present application, the polynucleotide encoding the SD being additionally fused to the N-terminus or C-terminus of a SOCS3 protein or an aMTD may be inserted into a cleavage site of restriction enzyme (Nde1, BamH1 and Sal1, etc.) within the multiple cloning sites (MCS) of a pET-28a(+) (Novagen, Darmstadt, Germany) or pET-26b(+) vector (Novagen, Darmstadt, Germany).

In still another embodiment disclosed in the present application, the polynucleotide encoding the iCP-SOCS3 recombinant protein may be cloned into a pET-28a(+) vector bearing a His-tag sequence so as to fuse six histidine residues to the N-terminus of the iCP-SOCS3 recombinant protein to allow easy purification.

According to one embodiment disclosed in the present application, the polynucleotide sequence may be a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 824, 826, 828 and 830.

The recombinant protein may be introduced into an appropriate host cell, e.g., a bacterial cell, a yeast cell, an insect cell, or a tissue culture cell. The recombinant protein may also be introduced into embryonic stem cells in order to generate a transgenic organism. Large numbers of suitable vectors and promoters are known to those skilled in the art and are commercially available for generating the recombinant protein.

Known methods may be used to construct vectors including the polynucleotide sequence according to one embodiment disclosed in the present application and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. For example, these techniques are described in the literatures [Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3d Edition, Cold Spring Harbor Laboratory, N.Y. (2001); and Ausubel et al., Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience, N.Y. (1989)].

Still another aspect disclosed in the present application provides a transformant transformed with the recombinant expression vector.

The transformation includes transfection, and refers to a process whereby a foreign (extracellular) DNA, with or without an accompanying material, enters into a host cell. The “transfected cell” refers to a cell into which the foreign DNA is introduced into the cell, and thus the cell harbors the foreign DNA. The DNA may be introduced into the cell so that a nucleic acid thereof may be integrated into the chromosome or replicable as an extrachromosomal element. The cell introduced with the foreign DNA, etc. is called a transformant.

As used herein, ‘introducing’ of a protein, a peptide, an organic compound into a cell may be used interchangeably with the expression of ‘carrying,’ ‘penetrating,’ ‘transporting,’ ‘delivering,’ ‘permeating’ or ‘passing.’

It is understood that the host cell refers to a eukaryotic or prokaryotic cell into which one or more DNAs or vectors are introduced, and refers not only to the particular subject cell but also to the progeny or potential progeny thereof. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

The host cells may be preferably bacterial cells, and as the bacterial cells, there are, in principle, no limitations. They may be eubacteria (gram-positive or gram-negative) or archaebacteria, as long as they allow genetic manipulation for insertion of a gene of interest, preferably for site-specific integration, and they may be cultured on a manufacturing scale. Preferably, the host cells may have the property to allow cultivation to high cell densities.

Examples of bacterial host cells that may be used in the preparation of the recombinant protein are E. coli (Lee, 1996; Hannig and Makrides, 1998), Bacillus subtilis, Pseudomonas fluorescens (Squires et al., 2004; Retallack et al., 2006) as well as various Corynebacterium (US 2006/0003404 A1) and Lactococcus lactis (Mierau et al., 2005) strains. Preferably, the host cells are Escherichia coli cells.

More preferably, the host cell may include an RNA polymerase capable of binding to a promoter regulating the gene of interest. The RNA polymerase may be endogenous or exogenous to the host cell.

Preferably, host cells with a foreign strong RNA polymerase may be used. For example, Escherichia coli strains engineered to carry a foreign RNA polymerase (e.g. like in the case of using a T7 promoter a T7-like RNA polymerase in the so-called “T7 strains”) integrated in their genome may be used. Examples of T7 strains, e.g. BL21(DE3), HMS174(DE3), and their derivatives or relatives (see Novagen, pET System manual, 11^(th) edition), may be widely used and commercially available. Preferably, BL21-CodonPlus (DE3)-RIL or BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) may be used. These strains are DE3 lysogens containing the T7 RNA polymerase gene under control of the lacUV5 promoter. Induction with IPTG allows production of T7 RNA polymerase which then directs the expression of the gene of interest under the control of the T7 promoter.

The host cell strains, E. coli BL21(DE3) or HMS174(DE3), which have received their genome-based T7 RNA polymerase via the phage DE3, are lysogenic. It is preferred that the T7 RNA polymerase contained in the host cell has been integrated by a method which avoids, or preferably excludes, the insertion of residual phage sequences in the host cell genome since lysogenic strains have the disadvantage to potentially exhibit lytic properties, leading to undesirable phage release and cell lysis.

Still another aspect disclosed in the present application provides a preparing method of the iCP-SOCS3 recombinant protein including preparing the recombinant expression vector; preparing the transformant using the recombinant expression vector; culturing the transformant; and recovering the recombinant protein expressed by culturing.

Culturing may be preferably in a mode that employs the addition of a feed medium, this mode being selected from the fed-batch mode, semi-continuous mode, or continuous mode, and the bacterial expression host cells may include a DNA construct, integrated in their genome, carrying the DNA sequence encoding the protein of interest under the control of a promoter that enables expression of said protein.

There are no limitations in the type of the culture medium. The culture medium may be semi-defined, i.e. containing complex media compounds (e.g. yeast extract, soy peptone, casamino acids), or it may be chemically defined, without any complex compounds. Preferably, a defined medium may be used. The defined media (also called minimal or synthetic media) are exclusively composed of chemically defined substances, i.e. carbon sources such as glucose or glycerol, salts, vitamins, and, in view of a possible strain auxotrophy, specific amino acids or other substances such as thiamine. Most preferably, glucose may be used as a carbon source. Usually, the carbon source of the feed medium serves as the growth-limiting component which controls the specific growth rate.

Host cells may be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents. The literature [Scopes, Protein Purification: Principles and Practice, New York: Springer-Verlag (1994)] describes a number of general methods for purifying recombinant (and non-recombinant) proteins. The methods may include, e.g., ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, selective precipitation, dialysis, and hydrophobic interaction chromatography. These methods may be adapted to devise a purification strategy for the cell permeable recombinant protein. If the cell permeable recombinant protein includes a purification handle, such as an epitope tag or a metal chelating sequence, affinity chromatography may be used to easily purify the protein.

The amount of the protein produced may be evaluated by detecting the advanced macromolecule transduction domain directly (e.g., using Western analysis) or indirectly (e.g., by assaying materials derived from the cells for specific DNA binding activity, such as by electrophoretic mobility shift assay). Proteins may be detected prior to purification, during any stage of purification, or after purification. In some implementations, purification or complete purification may not be necessary.

The iCP-SOCS3 recombinant proteins according to one embodiment disclosed in the present application are cell permeable proteins, and may be used as protein-based vaccines, particularly in the case where killed or attenuated whole organism vaccines are impractical.

The iCP-SOCS3 recombinant proteins according to one embodiment disclosed in the present application may be preferably used for the treating, preventing, or delaying the onset of, hepatocellular carcinoma. The cell permeable recombinant proteins may be delivered to the interior of the cell, eliminating the need to transfect or transform the cell with a recombinant vector. The cell permeable recombinant proteins may be used in vitro to investigate protein function or may be used to maintain cells in a desired state.

Still another aspect disclosed in the present application provides a composition including the iCP-SOCS3 Recombinant Protein as an active ingredient.

Still another aspect disclosed in the present application provides a pharmaceutical composition for treating, preventing, or delaying the onset of, hepatocellular carcinoma including the iCP-SOCS3 Recombinant Protein as an active ingredient; and a pharmaceutically acceptable carrier.

According to one embodiment disclosed in the present application, the iCP-SOCS3 Recombinant Protein may be used in a single agent, or in combination with one or more other anti-cancer agents.

Hepatocellular carcinoma (HCC) described herein include, but are not limited to, liver cell carcinomas, fibrolamellar variants of HCC, and mixed hepatocellular cholangiocarcinomas. In addition, the hepatocellular carcinoma may be early stage HCC, non-metastatic HCC, primary HCC, advanced HCC, locally advanced HCC, metastatic HCC, HCC in remission, recurrent HCC, HCC in an adjuvant setting, or HCC in a neoadjuvant setting.

Preferably, the composition may be for injectable (e.g. intraperitoneal, intravenous, and intra-arterial, etc.) and may include the active ingredient in an amount of 0.001 mg/kg to 1000 mg/kg, preferably 0.01 mg/kg to 100 mg/kg, more preferably 0.1 mg/kg to 50 mg/kg for human.

For examples, dosages per day normally fall within the range of about 0.001 to about 1000 mg/kg of body weight. In the treatment of adult humans, the range of about 0.1 to about 50 mg/kg/day, in single or divided dose, is especially preferred. However, it will be understood that the concentration of the iCP-SOCS3 recombinant protein actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the age, weight, and response of the individual patient, and the severity of the patient's symptoms, and therefore the above dosage ranges are not intended to limit the scope of the invention in any way. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several smaller doses for administration throughout the day.

Still another aspect disclosed in the present application provides use of the iCP-SOCS3 recombinant protein as a medicament for treating, preventing, or delaying the onset of, hepatocellular carcinoma.

Still another aspect disclosed in the present application provides a medicament including the iCP-SOCS3 recombinant protein.

Still another aspect disclosed in the present application provides use of the iCP-SOCS3 recombinant protein for the preparation of a medicament for treating, preventing, or delaying the onset of, hepatocellular carcinoma.

Still another aspect disclosed in the present application provides a method of treating, preventing, or delaying the onset of, hepatocellular carcinoma in a subject including identifying a subject in need of treating, preventing, or delaying the onset of, hepatocellular carcinoma; and administering to the subject a therapeutically effective amount of the iCP-SOCS3 recombinant protein.

In one embodiment disclosed in the present application, the subject may be preferably a mammal.

Preferably, the subject in need of treating, preventing, or delaying the onset of, hepatocellular carcinoma may be identified by any conventional diagnostic methods known in the art including ultrasound, CT scan, MRI, alpha-fetoprotein testing, des-gamma carboxyprothrombin screening, and biopsy, etc.

The people with hepatitis B or hepatitis C infection, or having cirrhosis acute and chronic hepatic porphyrias (acute intermittent porphyria, porphyria cutanea tarda, hereditary coproporphyria, variegate porphyria) and tyrosinemia type I may be the subject at an increased risk of for developing hepatocellular carcinoma. In addition, patients with a family history of hepatocellular carcinoma may also be identified for the subject in need of preventing or delaying the onset of hepatocellular carcinoma.

The pharmaceutical composition according to one embodiment disclosed in the present application may be prepared by using pharmaceutically suitable and physiologically acceptable additives, in addition to the active ingredient, and the additives may include excipients, disintegrants, sweeteners, binders, coating agents, blowing agents, lubricants, glidants, flavoring agents, etc.

For administration, the pharmaceutical composition may be preferably formulated by further including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredient.

Dosage forms of the pharmaceutical composition may include granules, powders, tablets, coated tablets, capsules, suppositories, liquid formulations, syrups, juice, suspensions, emulsions, drops, injectable liquid formulations, etc. For formulation of the composition into a tablet or capsule, for example, the active ingredient may be combined with any oral, non-toxic pharmaceutically acceptable inert carrier, such as ethanol, glycerol, water, etc. If desired or necessary, suitable binders, lubricants, disintegrants, and colorants may be additionally included as a mixture.

Examples of the suitable binder may include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Examples of the disintegrant may include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, etc. For formulation of the composition into a liquid preparation, a pharmaceutically acceptable carrier which is sterile and biocompatible may be used, such as saline, sterile water, a Ringer's solution, buffered saline, an albumin infusion solution, a dextrose solution, a maltodextrin solution, glycerol, and ethanol, and these materials may be used alone or in any combination thereof. If necessary, other common additives, such as antioxidants, buffers, bacteriostatic agents, etc., may be added. Further, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, and emulsions, or pills, capsules, granules, or tablets. Furthermore, the composition may be preferably formulated, depending upon diseases and ingredients, using any appropriate method known in the art, as disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton Pa.

Preferably, the treatment or treating mean improving or stabilizing the subject's condition or disease; or preventing or relieving the development or worsening of symptoms associated with the subject's condition or disease. The prevention, prophylaxis and preventive treatment are used herein as synonyms.

Preferably, the treating, preventing, or delaying the onset of, hepatocellular carcinoma may be any one or more of the following: alleviating one or more symptoms of HCC, delaying progressing of HCC, shrinking tumor size in HCC patient, inhibiting HCC tumor growth, prolonging overall survival, prolonging disease-free survival, prolonging time to HCC disease progression, preventing or delaying HCC tumor metastasis, reducing or eradiating preexisting HCC tumor metastasis, reducing incidence or burden of preexisting HCC tumor metastasis, preventing recurrence of HCC.

The subject and patient are used herein interchangeably. They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) that can be afflicted with or is susceptible to a disease or disorder but may or may not have the disease or disorder. In certain embodiments, the subject is a human being.

Preferably, the amount effective or effective amount is the amount of an active ingredient or a pharmaceutical composition disclosed herein that when administered to a subject for treating a disease, is sufficient to effect such treatment of the disease. Any improvement in the patient is considered sufficient to achieve treatment. An effective amount of an active ingredient or a pharmaceutical composition disclosed herein, used for the preventing, or delaying the onset of, hepatocellular carcinoma may vary depending upon the manner of administration, the age, body weight, and general health of the patient. Ultimately, the prescribers or researchers will decide the appropriate amount and dosage regimen.

In the treatment or prevention method according to one embodiment disclosed in the present application, the composition including the iCP-SOCS3 recombinant protein as an active ingredient may be administered in a common manner via oral, buccal, rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, percutaneous, topical, intraocular or subcutaneous route, more preferably via intraperitoneal, intravenous, or intra-arterial injection route.

Advantageous Effects

According to one aspect disclosed in the present application, development and establishment of improved cell-permeable SOCS3 recombinant protein, as therapeutics of hepatocellular carcinoma are provided. Because iCP-SOCS3 was designed based on endogenous proteins, it would be a safety anti-hepatocellular carcinoma drug without side-effect.

However, the effects of the disclosures in the present application are not limited to the above-mentioned effects, and another effects not mentioned will be clearly understood by those skilled in the art from the following description.

DESCRIPTION OF DRAWINGS

FIG. 1 shows Structure of aMTD- or rPeptide-Fused Recombinant Proteins. A schematic diagram of the His-tagged CRA recombinant proteins is illustrated and constructed according to the present invention. The his-tag for affinity purification (white), aMTD or rPeptide (gray) and cargo A (CRA, black) are shown.

FIG. 2a shows Construction of Expression Vectors for aMTDs- or rPeptide-Fused Recombinant Proteins. FIGS. 2b and 2c show the agarose gel electrophoresis analysis showing plasmid DNA fragments at 645 bp insert encoding aMTDs or rPeptide-fused CRA cloned into the pET28a(+) vector according to the present invention.

FIGS. 3a to 3d show Inducible Expression of aMTD- or rPeptide-Fused Recombinant Proteins. Expressed recombinant aMTD- or random peptide-fused CRA recombinant proteins were transformed in E. coli BL21 (DE3) strain. Expression of recombinant proteins in E. coli before (−) and after (+) induction with IPTG was monitored by SDS-PAGE, and stained with Coomassie blue.

FIGS. 4a and 4b show Purification of aMTD- or rPeptide-Fused Recombinant Proteins. Expressed recombinant proteins were purified by Ni²⁺ affinity chromatography under the natural condition. Purification of recombinant proteins displayed through SDS-PAGE analysis.

FIGS. 5a to 5u show Determination of aMTD-Mediated Cell-Permeability. Cell-permeability of a negative control (A: rP38) and reference hydrophobic CPPs (MTM12 and MTD85) are shown. The cell-permeability of each aMTD and/or rPeptide is visually compared to that of the cargo protein lacking peptide sequence (HCA). Gray shaded area represents untreated RAW 264.7 cells (vehicle); thin light gray line represents the cells treated with equal molar concentration of FITC (FITC only); dark thick line indicates the cells treated with FITC-his-tagged CRA protein (HCA); and the cells treated with the FITC-proteins (HMCA) fused to negative control (rP38), reference CPP (MTM12 or MTD85) or new hydrophobic CPP (aMTD) are shown with light thick line and indicated by arrows.

FIGS. 6a to 6c show Determination of rPeptide-Mediated Cell-Permeability. The cell-permeability of each aMTD and/or rPeptide was visually compared to that of the cargo protein lacking peptide sequence (HCA). Gray shaded area represents untreated RAW 264.7 cells (vehicle); thin light gray line represents the cells treated with equal molar concentration of FITC (FITC only); dark thick line indicates the cells treated with FITC-his-tagged CRA protein (HCA); and the cells treated with the FITC-proteins fused to rPeptides are shown with light thick line and indicated by arrows.

FIGS. 7a to 7k shows Visualized Cell-Permeability of aMTD-Fused Recombinant Proteins. NIH3T3 cells were treated with FITC-labeled protein (10 μM) fused to aMTD for 1 hour at 37. Cell-permeability of the proteins was visualized by laser scanning confocal microscopy (LSM700 version).

FIG. 8 shows Visualized Cell-Permeability of rPeptide-Fused Recombinant Proteins. Cell-permeability of rPeptide-fused recombinant proteins was visualized by laser scanning confocal microscopy (LSM700 version).

FIGS. 9a to 9c show Relative Cell-Permeability of aMTD-Fused Recombinant Proteins Compared to Negative Control (rP38). The FIG shows graphs comparing the cell-permeability of the recombinant proteins fused to aMTDs and a negative control (A: rP38).

FIGS. 10a to 10c show Relative Cell-Permeability of aMTD-Fused Recombinant Proteins Compared to Reference CPP (MTM12). The FIG shows graphs comparing the cell-permeability of the recombinant proteins fused to aMTDs and a reference CPP (MTM12).

FIGS. 11a to 11c show Relative Cell-Permeability of aMTD-Fused Recombinant Proteins Compared to Reference CPP (MTD85). The FIG shows graphs comparing the cell-permeability of the recombinant proteins fused to aMTDs and a reference CPP (MTD85).

FIG. 12 shows Relative Cell-Permeability of rPeptide-Mediated Recombinant Proteins Compared to Average that of aMTDs. The FIG shows graphs comparing the cell-permeability of the recombinant proteins fused to rPeptides and that (average value: aMTD AVE) of aMTDs.

FIGS. 13a and 13b show Association of Cell-Permeability with Amino Acid Composition in aMTD Sequences. These graphs display delivery potential (Geometric Mean) of aMTDs influenced with amino acid composition (A, I, V and L).

FIGS. 14a and 14b show Association of Cell-Permeability with Critical Factors in aMTDs. These graphs show the association of cell-permeability with critical factors [bending potential: proline position (PP), rigidity/flexibility: instability index (II), structural feature: aliphatic index (AI) and hydropathy: grand average of hydropathy (GRAVY)].

FIGS. 15a and 15b show Relative Relevance of aMTD-Mediated Cell-Permeability with Critical Factors. Cell-permeability of 10 high and 10 low ranked aMTDs in their delivery potential were examined for their association with the critical factors [bending potential: proline position (PP), rigidity/flexibility: instability index (II), structural feature: aliphatic index (AI) and hydropathy: grand average of hydropathy (GRAVY)].

FIG. 16 shows Relative Relevance of rPeptide-Mediated Cell-Permeability with Hydropathy Range (GRAVY). This graph and a chart illustrate relative relevance of rPeptide-mediated cell-permeability with its hydropathy range (GRAVY).

FIG. 17 shows a structure of iCP-SOCS3 recombinant protein designed according to example 6-1.

FIG. 18 shows the agarose gel electrophoresis analysis showing plasmid DNA fragments insert encoding His-SOCS3-SDB (HS3B), His-aMTD₁₆₅-SOCS3-SDB (HM₁₆₅S3B), His-aMTD₁₆₅-SOCS3-SDC (HM₁₆₅S3C), His-aMTD₁₆₅-SOCS3-SDD (HM₁₆₅S3D), His-aMTD₁₆₅-SOCS3-SDE (HM₁₆₅S3E) cloned into the pET28a (+) vector according to example 6-1.

FIG. 19 shows inducible expression and purification of iCP-SOCS3 recombinant protein in E. coli according to example 6-2 and improvement of solubility/yield of iCP-SOCS3 recombinant protein by fusing aMTD/SD according to example 6-3.

FIG. 20 shows aMTD-Mediated cell-permeability of SOCS3 recombinant proteins in RAW 264.7 cells according to example 7-1

FIG. 21 shows aMTD-Mediated intracellular delivery and localization of SOCS3 Recombinant Proteins in NIH3T3 cells according to example 7-1.

FIG. 22 shows systemic delivery of aMTD/SD-fused SOCS3 recombinant proteins in vivo according to example 7-2.

FIG. 23 shows inhibition of IFN-γ-induced STAT phosphorylation by iCP-SOCS3 recombinant protein according to example 8-1.

FIG. 24 shows inhibition of LPS-induced cytokines secretion by iCP-SOCS3 recombinant protein according to example 8-2.

FIG. 25 shows the structures of SOCS3 recombinant protein lacking aMTD prepared as a negative control according to example 9.

FIG. 26 shows expression, purification, and solubility/yield of HS3 (lacking aMTD and SD) and HS3B (lacking aMTD) determined according to example 6-3.

FIG. 27 shows the agarose gel electrophoresis analysis showing plasmid DNA fragments insert encoding His-aMTD#-SOCS3-SDB (HM_(#)S3B) and His-rP#-SOCS3-SDB cloned into the pET28a (+) vector according to example 10.

FIGS. 28a and 28b show expression, purification, and solubility/yield of His-aMTD#-SOCS3-SDB (HM_(#)S3B) determined according to example 10.

FIG. 29 shows expression, purification, and solubility/yield of His-rP#-SOCS3-SDB (HrP_(#)S3B) determined according to example 10.

FIG. 30 shows solubility/yield of His-aMTD#-SOCS3-SDB (HM_(#)S3B) determined according to example 10.

FIG. 31 show aMTD-mediated cell-permeability. The cell-permeability of each SOCS3 recombinant protein fused with SD and various aMTD is visually compared to that of the cargo protein lacking CPP (HS3B) or lacking CPP and SD (HS3). Gray shaded area represents untreated E. coli cells (diluent); green line represents the cells treated with equal molar concentration of FITC (FITC only); black line indicates the cells treated with FITC-his-SOCS protein (FITC-HS3); blue line indicates the cells treated with FITC-his-SOCS-SDB protein (FITC-HS3B) purple line indicates the cells treated with FITC-his-aMTD_(#)-SOCS-SDB protein (FITC-HM_(#)S3B).

FIG. 32 shows relative cell-permeability of His-aMTD_(#)-SOCS3-SDB-Fused recombinant proteins Compared to control (Vehicle, FITC only, HS3 and HS3B).

FIG. 33 shows random Peptide-Mediated cell-permeability. The cell-permeability of each SOCS3 recombinant protein fused with SDB and aMTD₁₆₅ or various rP is visually compared to that of the cargo protein lacking CPP (HS3B) or lacking CPP and SD (HS3). Gray shaded area represents untreated E. coli cells (diluent); green line represents the cells treated with equal molar concentration of FITC (FITC only); black line indicates the cells treated with FITC-his-SOCS protein (FITC-HS3); blue line indicates the cells treated with FITC-his-SOCS-SDB protein (FITC-HS3B) and purple line indicates the cells treated with FITC-his-rPeptide_(#)-SOCS-SDB protein (FITC-HrP_(#)S3B).

FIG. 34 shows relative cell-permeability of His-rP_(#)-SOCS3-SDB-Fused recombinant proteins Compared to control (Vehicle, FITC only, HS3 and HS3B).

FIG. 35 shows apoptotic cells analysis according to example 11-1.

FIG. 36 shows induction of apoptosis by iCP-SOCS3 recombinant proteins according to example 11-2.

FIG. 37 shows cell migration inhibition by iCP-SOCS3 recombinant protein according to example 11-3.

FIG. 38 shows solubility/yield, permeability and biological activity of His-aMTD#-SOCS3-SDB (HM_(#)S3B) determined according to example 10 to 11-3.

FIG. 39 shows expression, purification, and solubility/yield of M₁₆₅S3SB (lacking his-tag) determined according to example 12-1.

FIG. 40 shows cell-permeability of SOCS3 recombinant proteins (lacking his-tag) in RAW 264.7 cells according to example 12-2.

FIG. 41 shows Annexin V analysis according to example 12-3.

FIG. 42 shows cell migration inhibition (bottom) by iCP-SOCS3 recombinant protein according to example 12-3.

FIG. 43 shows a structure of iCP-SOCS3 recombinant protein (His-aMTD₁₆₅-SOCS3-SDB′) constructed according to example 12-4.

FIG. 44 shows expression, purification, and solubility/yield of HM₁₆₅S3SB and HM₁₆₅S3SB′ determined according to example 12-4.

FIG. 45 shows aMTD-Mediated cell-permeability of iCP-SOCS3 recombinant proteins (HM₁₆₅S3B and HM₁₆₅S3B′(V28L)) in RAW 264.7 cells according to example 12-5.

FIG. 46 shows antiproliferative activity of iCP-SOCS3 recombinant proteins (HM₁₆₅S3B and HM₁₆₅S3B′(V28L)) according to example 12-6.

FIG. 47 shows induction of apoptosis by iCP-SOCS3 recombinant proteins (HM₁₆₅S3B and HM₁₆₅S3B′(V28L)) according to example 12-6.

FIG. 48 shows cell migration inhibition by SOCS3 recombinant proteins (HM₁₆₅S3B and HM₁₆₅S3B′(V28L)) according to example 12-6.

FIG. 49a shows a structure of iCP-SOCS3 recombinant proteins (top) and agarose gel electrophoresis analysis (bottom) according to example 12-7 and FIG. 49b shows inducible expressions and purifications of iCP-SOCS3 recombinant protein in E. coli (bottom) according to example 12-7.

FIG. 50 shows inhibition of IFN-γ-induced STAT phosphorylation by iCP-SOCS3 recombinant protein according to example 13.

FIG. 51 shows effect of treating EDTA (FIG. 51A) and proteinase K (FIG. 51B) on aMTD-mediated SOCS3 protein uptake into cells according to example 14-2.

FIG. 52 shows effect of treating taxol (FIG. 52 A) and antimycin (FIG. 52 B) on aMTD-mediated SOCS3 protein uptake into cells according to example 14-2.

FIG. 53 shows effect of temperature on aMTD-mediated SOCS3 protein uptake into cells according to example 14-2.

FIG. 54 shows aMTD-mediated cell-to-cell delivery assessed according to example 14-2.

FIG. 55 shows bioavailability of iCP-SOCS3 recombinant protein in PBMC, splenocytes and hepatocytes analyzed by fluorescence microscopy according to example 15.

FIG. 56 shows biodistribution of iCP-SOCS3 recombinant protein in pancreas tissues analyzed by confocal microscope according to example 15.

FIG. 57 shows aMTD-Mediated cell-permeability of SOCS3 recombinant proteins in HepG2 cells according to example 16.

FIG. 58 shows systemic delivery of aMTD/SD-fused SOCS3 recombinant proteins to liver according to example 16.

FIG. 59 shows methylation and unmethylation level of endogenous SOCS3 in cell line analyzed by the agarose gel electrophoresis according to example 17-1.

FIG. 60 shows expression level of endogenous SOCS3 mRNA in cell line analyzed by the agarose gel electrophoresis according to example 17-2.

FIG. 61 shows expression level of SOCS3 gene, and phosphorylation of JAK1 and JAK2 in cell line analyzed by the agarose gel electrophoresis according to example 17-3.

FIG. 62 shows antiproliferative activity of iCP-SOCS3 recombinant protein according to example 18.

FIG. 63 shows cell migration inhibition activity by iCP-SOCS3 recombinant protein according to example 19.

FIGS. 64a to 64c show transwell migration inhibition activity by iCP-SOCS3 recombinant protein in HepG2 cells (FIG. 64a ), Huh7 cells (FIG. 64b ) and SK-HEP1 cells (FIG. 64c ) according to example 19.

FIG. 65 shows decrease in invasion by iCP-SOCS3 recombinant protein in HepG2 cells analyzed according to example 19.

FIG. 66 shows induction of apoptosis of hepatic cancer cells by iCP-SOCS3 recombinant proteins in a dose-dependent manner assessed by Annexin V staining according to example 20-1.

FIGS. 67 and 68 show induction of apoptosis in normal Chang liver cells (FIG. 67) and in HCCs (FIG. 68) by iCP-SOCS3 recombinant proteins assessed by Annexin V staining according to example 20-2.

FIGS. 69 and 70 show induction of apoptosis in normal Chang liver cells (FIG. 69) and in Huh7 (FIG. 70) by iCP-SOCS3 recombinant proteins analyzed by TUNEL assay according to example 21.

FIGS. 71 and 72 show inhibition of cell cycle progression in normal liver cells (FIG. 71) and in HCCs (FIG. 72) by iCP-SOCS3 recombinant proteins assessed by flow cytometric analysis according to example 22.

FIG. 73 shows down regulation of expression of apoptosis by iCP-SOCS3 recombinant proteins in HCCs determined by immunoblotting according to example 23.

FIG. 74 shows suppression of the tumor growth by iCP-SOCS3 recombinant proteins in Hep3B2.1-7 assessed according to example 24-1.

FIG. 75 shows suppression of the tumor growth by iCP-SOCS3 recombinant proteins in HepG2 assessed according to example 24-2.

FIG. 76 shows suppression of the tumor growth by iCP-SOCS3 recombinant proteins in HepG2 by varying dose and time according to example 24-3.

FIG. 77 shows suppression of the tumor growth by iCP-SOCS3 recombinant proteins and cisplatin in HepG2 assessed according to example 24-4.

FIG. 78 shows humanized SDB domain according to example 12-4.

FIG. 79 shows sequences of amino acid and nucleotide of basic CPP, and primers used in example 6-4.

FIG. 80 shows structure, expression, purification and solubility/yield of aMTD/SD-fused SOCS3 recombinant protein and basic CPP/SD-fused SOCS3 recombinant protein analyzed according to example 6-4.

FIG. 81 shows comparison of cell-permeability between aMTD/SD fused SOCS3 recombinant proteins and basic CPP/SD-fused in RAW 264.7 cells according to example 7-1-2.

FIG. 82 shows comparison of tissue-permeability between aMTD/SD fused SOCS3 recombinant proteins and basic CPP/SD-fused in various tissues of ICR mice according to example 7-2-2.

FIG. 83 shows effect of treating proteinase K (A) and Taxol (B) on aMTD (or basic CPP)-mediated SOCS3 protein uptake into cells according to example 14.

FIGS. 84 and 85 show aMTD (or basic CPP)-mediated cell-to-cell delivery (FIG. 84) and cell-to-cell function (FIG. 85) assessed according to example 14.

FIGS. 86a and 86b show dose-dependency of cell-permeability of iCP-SOCS3 recombinant proteins analyzed according to example 14-1.

FIG. 87 shows time-dependency of cell-permeability of the iCP-SOCS3 recombinant proteins analyzed according to example 14-1.

FIG. 88 shows the established pancreatic cancer xenograft model (A) and tumor targeting of the iCP-SOCS3 recombinant proteins (B) assessed according to example 16-1.

MODE FOR INVENTION 1. Analysis of Reference Hydrophobic CPPs to Identify ‘Critical Factors’ for Development of Advanced MTDs

Previously reported MTDs were selected from a screen of more than 1,500 signal peptide sequences. Although the MTDs that have been developed did not have a common sequence or sequence motif, they were all derived from the hydrophobic (H) regions of signal sequences (HRSSs) that also lack common sequences or motifs except their hydrophobicity and the tendency to adopt alpha-helical conformations. The wide variation in H-region sequences may reflect prior evolution for proteins with membrane translocating activity and subsequent adaptation to the SRP/Sec61 machinery, which utilizes a methionine-rich signal peptide binding pocket in SRP to accommodate a wide-variety of signal peptide sequences.

Previously described hydrophobic CPPs (e.g. MTS/MTM and MTD) were derived from the hydrophobic regions present in the signal peptides of secreted and cell surface proteins. The prior art consists first, of ad hoc use of H-region sequences (MTS/MTM), and second, of H-region sequences (with and without modification) with highest CPP activity selected from a screen of 1,500 signal sequences (MTM). Second prior art, the modified H-region derived hydrophobic CPP sequences had advanced in diversity with multiple number of available sequences apart from MTS/MTM derived from fibroblast growth factor (FGF) 4. However, the number of MTDs that could be modified from naturally occurring secreted proteins are somewhat limited. Because there is no set of rules in determining their cell-permeability, no prediction for the cell-permeability of modified MTD sequences can be made before testing them.

The hydrophobic CPPs, like the signal peptides from which they originated, did not conform to a consensus sequence, and they had adverse effects on protein solubility when incorporated into protein cargo. We therefore set out to identify optimal sequence and structural determinants, namely critical factors (CFs), to design new hydrophobic CPPs with enhanced ability to deliver macromolecule cargoes including proteins into the cells and tissues while maintaining protein solubility. These newly developed CPPs, advanced macromolecule transduction domains (aMTDs) allowed almost infinite number of possible designs that could be designed and developed based on the critical factors. Also, their cell-permeability could be predicted by their character analysis before conducting any in vitro and/or in vivo experiments. These critical factors below have been developed by analyzing all published reference hydrophobic CPPs.

1-1. Analysis of Hydrophobic CPPs

Seventeen different hydrophobic CPPs (Table 1) published from 1995 to 2014 (Table 2) were selected. After physiological and chemical properties of selected hydrophobic CPPs were analyzed, 11 different characteristics that may be associated with cell-permeability have been chosen for further analysis. These 11 characteristics are as follows: sequence, amino acid length, molecular weight, pI value, bending potential, rigidity/flexibility, structural feature, hydropathy, residue structure, amino acid composition and secondary structure of the sequences (Table 3).

Table 1 shows the summary of published hydrophobic Cell-Penetrating Peptides which were chosen.

TABLE 1 # Peptides Origin Protein Ref. 1 MTM Homo sapiens NP_001998 Kaposi fibroblast growth factor (K-FGF) 1 2 MTS Homo sapiens NP_001998 Kaposi fibroblast growth factor (K-FGF) 2 3 MTD10 Streptomyces coelicolor NP_625021 Glycosyl hydrolase 8 4 MTD13 Streptomyces coelicolor NP_639877 Putative secreted protein 3 5 MTD47 Streptomyces coelicolor NP_627512 Secreted protein 4 6 MTD56 Homo sapiens P23274 Peptidyl-prolyl cis-trans isomerase B precursor 5 7 MTD73 Drosophila melanogaster AAA17887 Spatzle (spz) protein 5 8 MTD77 Homo sapiens NP_003231 Kaposi fibroblast growth factor (K-FGF) 6 9 MTD84 Phytophthora cactorum AAK63068 Phytotoxic protein PcF precusor 4 10 MTD85 Streptomyces coelicolor NP_629842 Peptide transport system peptide binding 7 protein 11 MTD86 Streptomyces coelicolor NP_629842 Peptide transport system secreted peptide 7 binding protein 12 MTD103 Homo sapiens TMBV19 domain Family member B 8 13 MTD132 Streptomyces coelicolor NP_628377 P60-family secreted protein 4 14 MTD151 Streptomyces coelicolor NP_630126 Secreted chitinase 8 15 MTD173 Streptomyces coelicolor NP_624384 Secreted protein 4 16 MTD174 Streptomyces coelicolor NP_733505 Large, multifunctional secreted protein 8 17 MTD181 Neisseria meningitidis Z2491 CAB84257.1 Putative secreted protein 4

Table 2 summarizes reference information

TABLE 2 References # Title Journal Year Vol Issue Page 1 Inhibition of Nuclear Translocation of Transcription JOURNAL OF 1995 270 24 14255 Factor NF-kB by a Synthetic peptide Containing a BIOLOGICAL Cell Membrane-permeable Motif and Nuclear CHEMISTRY Localization Sequence 2 Epigenetic Regulation of Gene Structure and NATURE 2001 19 10 929 Function with a Cell-Permeable Cre Recombinase BIOTECHNOLOGY 3 Cell Permeable NM23 Blocks the Maintenance and CANCER 2011 71 23 7216 Progression of Established Pulmonary Metastasis RESEARCH 4 Antitumor Activity of Cell-Permeable p18INK4c MOLECULAR 2012 20 8 1540 With Enhanced Membrane and Tissue Penetration THERAPY 5 Antitumor Activity of Cell Permeable RUNX3 Protein CLINICAL 2012 19 3 680 in Gastric Cancer Cells CANCER RESEARCH 6 The Effect of Intracellular Protein Delivery on the Anti- BIOMATERIALS 2013 34 26 6261 Tumor Activity of Recombinant Human Endostatin 7 Partial Somatic to Stem Cell Transformations Induced SCIENTIFIC 2014 4 10 4361 By Cell Permeable Reprogramming Factors REPORTS 8 Cell-Permeable Parkin Proteins Suppress Parkinson PLOS ONE 2014 9 7 17 Disease Associated Phenotypes in Cultured Cells and Animals

Table 3 shows characteristics of published hydrophobic Cell-Penetrating Peptides (A) which were analyzed.

TABLE 3 Rigidity/ Structural Flexibility Feature Molecular Bending (Instability (Aliphatic # Peptides Sequence Length Weight pI Potential Index: II) Index: Al)  1 MTM AAVALLPAVLLALLAP 16 1,515.9 5.6 Bending 45.5 220.0  2 MTS AAVLLPVLLAAP 12 1,147.4 5.6 Bending 57.3 211.7  3 MTD10 LGGAVVAAPVAAAVAP 16 1,333.5 5.5 Bending 47.9 140.6  4 MTD13 LAAAALAVLPL 11 1,022.3 5.5 Bending 26.6 213.6  5 MTD47 AAAVPVLVAA 10 881.0 5.6 Bending 47.5 176.0  6 MTD56 VLLAAALIA 9 854.1 5.5 No- 8.9 250.0 Bending  7 MTD73 PVLLLLA 7 737.9 6.0 No- 36.1 278.6 Bending  8 MTD77 AVALLILAV 9 882.1 5.6 No- 30.3 271.1 Bending  9 MTD84 AVALVAVVAVA 11 982.2 5.6 No- 9.1 212.7 Bending 10 MTD85 LLAAAAALLLA 11 1,010.2 5.5 Bending 9.1 231.8 11 MTD86 LLAAAAALLUk 11 1,010.2 5.5 Bending 9.1 231.8 12 MTD103 LALPVLLLA 9 922.2 5.5 Bending 51.7 271.1 13 MTD132 AVVVPAIVLAAP 12 1,119.4 5.6 Bending 50.3 195.0 14 MTD151 AAAPVAAVP 9 1,031.4 5.5 Bending 73.1 120.0 15 MTD173 AVIPILAVP 9 892.1 5.6 Bending 48.5 216.7 16 MTD174 LILLLPAVALP 12 1,011.8 5.5 Bending 79.1 257.3 17 MTD181 AVLLLPAAA 9 838.0 5.6 Bending 51.7 206.7 AVE 10.8 ± 2.4 1,011 ± 189.6 5.6 ± 0.1 Proline 40.1 ± 21.9 217.9 ± 43.6 Presence A/a Hydropathy Residue Composition Secondary # (GRAVY) Structure A V L I P G Structure Cargo Ref.  1 2.4 Aliphatic 6 2 6 0 2 0 Helix p50 1 Ring  2 2.3 — 4 2 4 0 2 0 No-Helix CRE 2  3 1.8 — 7 4 1 0 2 2 Helix Parkin 8  4 2.4 — 5 1 4 0 1 0 No-Helix RUNX3 3  5 2.4 — 5 3 1 0 1 0 No-Helix CMYC 4  6 3.0 — 4 1 3 1 0 0 Helix ES 5  7 2.8 — 1 1 4 0 1 0 Helix ES 5  8 3.3 — 3 2 3 1 0 0 Helix NM23 6  9 3.1 — 5 5 1 0 0 0 Helix OCT4 4 10 2.7 — 6 0 5 0 0 0 No-Helix RUNX3 7 11 2.7 — 6 0 5 0 0 0 No-Helix SOX2 7 12 2.8 — 2 1 5 0 1 0 Helix p18 8 13 2.4 — 4 4 1 1 2 0 No-Helix LIN28 4 14 1.6 — No-Helix Parkin 8 15 2.4 — 2 2 1 2 2 0 Helix KLF4 4 16 2.6 — Helix Partin 8 17 2.4 — 4 1 3 0 1 0 No-Helix SOX2 4 2.5 ± 0.4

Two peptide/protein analysis programs were used (ExPasy: SoSui: http://harrier.nagahama-i-bio.ac.jp/sosui/sosui_submit.html) to determine various indexes and structural features of the peptide sequences and to design new sequence. Followings are important factors analyzed.

1-2. Characteristics of Analyzed Peptides: Length, Molecular Weight and pI Value

Average length, molecular weight and pl value of the peptides analyzed were 10.8±2.4, 1.011±189.6 and 5.6±0.1, respectively (Table 4)

Table 4 summarizes Critical Factors (CFs) of published hydrophobic Cell-Penetrating Peptides (A) which were analyzed.

TABLE 4 Length: 10.8 ± 2.4 Molecular Weight: 1,011 ± 189.6 pl: 5.6 ± 0.1 Bending Potential (BP): Proline presences in the middle and/or the end of peptides, or No Proline. Instability Index (II): 40.1 ± 21.9 Residue Structure & Aliphatic Index (AI): 217.9 ± 43.6 Hydropathy (GRAVY): 2.5 ± 0.4 Aliphatic Ring: Non-polar hydrophobic & aliphatic amino acid (A, V, L, I). Secondary Structure: α-Helix is favored but not required.

1-3. Characteristics of Analyzed Peptides: Bending Potential—Proline Position (PP)

Bending potential (bending or no-bending) was determined based on the fact whether proline (P) exists and/or where the amino acid(s) providing bending potential to the peptide in recombinant protein is/are located. Proline differs from the other common amino acids in that its side chain is bonded to the backbone nitrogen atom as well as the alpha-carbon atom. The resulting cyclic structure markedly influences protein architecture which is often found in the bends of folded peptide/protein chain.

Eleven out of 17 were determined as ‘Bending’ peptide which means that proline is present in the middle of sequence for peptide bending and/or located at the end of the peptide for protein bending. As indicated above, peptide sequences could penetrate the plasma membrane in a “bent” configuration. Therefore, bending or no-bending potential is considered as one of the critical factors for the improvement of current hydrophobic CPPs.

1-4. Characteristics of Analyzed Peptides: Rigidity/Flexibility—Instability Index (II)

Since one of the crucial structural features of any peptide is based on the fact whether the motif is rigid or flexible, which is an intact physicochemical characteristic of the peptide sequence, instability index (II) of the sequence was determined. The index value representing rigidity/flexibility of the peptide was extremely varied (8.9-79.1), but average value was 40.1±21.9 which suggested that the peptide should be somehow flexible, but not too much rigid or flexible (Table 3).

1-5. Characteristics of Analyzed Peptides: Structural Features—Structural Feature (Aliphatic Index: AI) and Hydropathy (Grand Average of Hydropathy: GRAVY)

Alanine (V), valine (V), leucine (L) and isoleucine (I) contain aliphatic side chain and are hydrophobic—that is, they have an aversion to water and like to cluster. These amino acids having hydrophobicity and aliphatic residue enable them to pack together to form compact structure with few holes. Analyzed peptide sequence showed that all composing amino acids were hydrophobic (A, V, L and I) except glycine (G) in only one out of 17 (MTD10—Table 3) and aliphatic (A, V, L, I, and P). Their hydropathic index (Grand Average of Hydropathy: GRAVY) and aliphatic index (AI) were 2.5±0.4 and 217.9±43.6, respectively. Their amino acid composition is also indicated in the Table 3.

1-6. Characteristics of Analyzed Peptides: Secondary Structure (Helicity)

As explained above, the CPP sequences may be supposed to penetrate the plasma membrane directly after inserting into the membranes in a “bent” configuration with hydrophobic sequences having α-helical conformation. In addition, our analysis strongly indicated that bending potential was crucial for membrane penetration. Therefore, structural analysis of the peptides was conducted to determine whether the sequences were to form helix or not. Nine peptides were helix and eight were not (Table 3). It seems to suggest that helix structure may not be required.

1-7. Determination of Critical Factors (CFs)

In the 11 characteristics analyzed, the following 6 are selected namely “Critical Factors” for the development of new hydrophobic CPPs—advanced MTDs: amino acid length, bending potential (proline presence and location), rigidity/flexibility (instability index: II), structural feature (aliphatic index: AI), hydropathy (GRAVY) and amino acid composition/residue structure (hydrophobic and aliphatic A/a) (Table 3 and Table 4).

2. Analysis of Selected Hydrophobic CPPs to Optimize ‘Critical Factors’

Since the analyzed data of the 17 different hydrophobic CPPs (analysis A, Table 3 and 4) previously developed during the past 2 decades showed high variation and were hard to make common- or consensus-features, analysis B (Table 5 and 6) and C (Table 7 and 8) were also conducted to optimize the critical factors for better design of improved CPPs-aMTDs. Therefore, 17 hydrophobic CPPs have been grouped into two groups and analyzed the groups for their characteristics in relation to the cell permeable property. The critical factors have been optimized by comparing and contrasting the analytical data of the groups and determining the common homologous features that may be critical for the cell permeable property.

2-1. Selective Analysis (B) of Peptides Used to Biologically Active Cargo Protein for In Vivo

In analysis B, eight CPPs were used with each biologically active cargo in vivo. Length was 11±3.2, but 3 out of 8 CPPs possessed little bending potential. Rigidity/Flexibility (instability index: II) was 41±15, but removing one [MTD85: rigid, with minimal II (9.1)] of the peptides increased the overall instability index to 45.6±9.3. This suggested that higher flexibility (40 or higher II) is potentially be better. All other characteristics of the 8 CPPs were similar to the analysis A, including structural feature and hydropathy (Table 5 and 6)

Table 5 shows characteristics of published hydrophobic Cell-Penetrating Peptides (B): selected CPPs that were used to each cargo in vivo.

TABLE 5 Rigidity/ Structural Flexibility Feature Molecular Bending (Instability (Aliphatic # Peptides Sequence Length Weight pI Potential Index: II) Index: Al) 1 MTM AAVALLPAVLLALLAP 16 1,515.9 5.6 Bending 45.5 220.0 2 MTS AAVLLPVLLAAP 12 1,147.4 5.6 Bending 57.3 211.7 3 MTD10 LGGAVVAAPVAAAVAP 16 1,333.5 5.5 Bending 47.9 140.6 4 MTD73 PVLLLLA 7 737.9 6.0 No- 36.1 278.6 Bending 5 MTD77 AVALLILAV 9 882.1 5.6 No- 30.3 271.1 Bending 6 MTD85 LLAAAAALLLA 11 1,010.2 5.5 No- 9.1* 231.8 Bending 7 MTD103 LALPVLLLA 9 922.2 5.5 Bending 51.7 271.1 8 MTD132 AVVVPAIVLAAP 12 1,119.4 5.6 Bending 50.3 195.0 AVE 11 ± 3.2 1,083 ± 252 5.6 ± 0.1 Proline 41 ± 15 227 ± 47 Presence A/a Hydropathy Residue Composition Secondary # (GRAVY) Structure A V L I P G Structure Cargo Ref. 1 2.4 Aliphatic 6 2 6 0 2 0 Helix p50 1 Ring 2 2.3 — 4 2 4 0 2 0 No-Helix CRE 2 3 1.8 — 7 4 1 0 2 2 Helix Parkin 8 4 2.8 — 1 1 4 0 1 0 Helix ES 6 5 3.3 — 3 2 3 1 0 0 Helix NM23 3 6 2.7 — 6 0 5 0 0 0 No-Helix RUNX3 5 7 2.8 — 2 1 5 0 1 0 Helix p18 4 8 2.4 — 4 4 1 1 2 0 No-Helix LIN28 7 2.5 ± 0.4 *Removing the MTD85 increases II to 45.6 ± 93

Table 6 shows summarized Critical Factors of published hydrophobic Cell-Penetrating Peptides (B).

TABLE 6 Length: 11 ± 3.2 Molecular Weight 1,083 ± 252 pl: 5.6 ± 0.1 Bending Potential (BP): Proline presences in the middle and/or the end of peptides, or No Proline. Instability Index (II): 41.0 ± 15 (^(▪) Removing the MTD85 increases II to 45.6 ± 9.3) Residue Structure & Aliphatic Index (AI): 227 ± 47 Hydropathy (GRAVY): 2.5 ± 0.4 Aliphatic Ring: Non-polar hydrophobic & aliphatic amino acid (A, V, L, I). Secondary Structure: α-Helix is favored but not required.

2-2. Selective Analysis (C) of Peptides that Provided Bending Potential and Higher Flexibility

To optimize the ‘Common Range and/or Consensus Feature of Critical Factor’ for the practical design of aMTDs and the random peptides (rPs or rPeptides), which were to prove that the ‘Critical Factors’ determined in the analysis A, B and C were correct to improve the current problems of hydrophobic CPPs—protein aggregation, low solubility/yield, and poor cell-/tissue-permeability of the recombinant proteins fused to the MTS/MTM or MTD, and non-common sequence and non-homologous structure of the peptides, empirically selected peptides were analyzed for their structural features and physicochemical factor indexes.

Hydrophobic CPPs which did not have a bending potential, rigid or too much flexible sequences (too much low or too much high Instability Index), or too low or too high hydrophobic CPPs were unselected, but secondary structure was not considered because helix structure of sequence was not required.

In analysis C, eight selected CPP sequences that could provide a bending potential and higher flexibility were finally analyzed (Table 7 and 8). Common amino acid length is 12 (11.6±3.0). Proline is presence in the middle of and/or the end of sequence. Rigidity/Flexibility (II) is 45.5-57.3 (Avg: 50.1±3.6). AI and GRAVY representing structural feature and hydrophobicity of the peptide are 204.7±37.5 and 2.4±0.3, respectively. All peptides are consisted with hydrophobic and aliphatic amino acids (A, V, L, I, and P). Therefore, analysis C was chosen as a standard for the new design of new hydrophobic CPPs-aMTDs.

Table 7 shows characteristics of published hydrophobic Cell-Penetrating Peptides (C): selected CPPs that provided bending potential and higher flexibility.

TABLE 7 Rigidity/ Structural Flexibility Feature Molecular Bending (Instability (Aliphatic # Peptides Sequence Length Weight pI Potential Index: II) Index: Al) 1 MTM AAVALLPAVLLALLAP 16 1515.9 5.6 Bending 45.5 220.0 2 MTS AAVLLPVLLAAP 12 1147.4 5.6 Bending 57.3 211.7 3 MTD10 LGGAVVAAPVAAAVAP 16 1333.5 5.5 Bending 47.9 140.6 4 MTD47 AAAVPVLVAA 10 881.0 5.6 Bending 47.5 176.0 5 MTD103 LALPVLLLA 9 922.2 5.5 Bending 51.7 271.1 6 MTD132 AVVVPAIVLAAP 12 1119.4 5.6 Bending 50.3 195.0 7 MTD173 AVIPILAVP 9 892.1 5.6 Bending 48.5 216.7 8 MTD181 AVLLLPAAA 9 838.0 5.6 Bending 51.7 206.7 AVE 11.6 ± 3.0 1081.2 ± 244.6 5.6 ± 0.1 Proline 50.1 ± 3.6 204.7 ± 37.5 Presence A/a Hydropathy Residue Composition Secondary # (GRAVY) Structure A V L I P G Structure Cargo Ref. 1 2.4 Aliphatic 6 2 6 0 2 0 Helix p50 1 Ring 2 2.3 — 4 2 4 0 2 0 No-Helix CRE 2 3 1.8 — 7 4 1 0 2 2 Helix Parkin 8 4 2.4 — 5 3 1 0 1 0 No-Helix CMYC 4 5 2.8 — 2 1 5 0 1 0 Helix p18 8 6 2.4 — 4 4 1 1 2 0 No-Helix LIN28 4 7 2.4 — 2 2 1 2 2 0 Helix KLE4 4 8 2.4 — 4 1 3 0 1 0 No-Helix SOX2 4 2.4 ± 0.3

Table 8 shows summarized Critical Factors of published hydrophobic Cell-Penetrating Peptides (C)

TABLE 8 Length: 11.6 ± 3.0 Molecular Weight 1,081.2 ± 224.6 pl: 5.6 ± 0.1 Bending Potential (BP) Proline presences in the middle and/or the end of peptides. Instability Index (II): 50.1 ± 3.6 Residue Structure & Aliphatic Index (AI): 204.7 ± 37.5 Hydropathy (GRAVY): 2.4 ± 0.3 Aliphatic Ring: Non-polar hydrophobic & aliphatic amino acid (A, V, L, I). Secondary Structure: α-Helix is favored but not required.

3. New Design of Improved Hydrophobic CPPs-aMTDs Based on the Optimized Critical Factors

3-1. Determination of Common Sequence and/or Common Homologous Structure

As mentioned above, H-regions of signal sequence (HRSS)-derived CPPs (MTS/MTM and MTD) do not have a common sequence, sequence motif, and/or common-structural homologous feature. In this invention, the aim is to develop improved hydrophobic CPPs formatted in the common sequence- and structural-motif which satisfy newly determined ‘Critical Factors’ to have ‘Common Function,’ namely, to facilitate protein translocation across the membrane with similar mechanism to the analyzed reference CPPs. Based on the analysis A, B and C, the common homologous features have been analyzed to determine the critical factors that influence the cell-permeability. The range value of each critical factor has been determined to include the analyzed index of each critical factor from analysis A, B and C to design novel aMTDs (Table 9). These features have been confirmed experimentally with newly designed aMTDs in their cell-permeability.

Table 9 shows comparison the range/feature of each Critical Factor between the value of analyzed CPPs and the value determined for new design of novel aMTDs sequences

TABLE 9 Summarized Critical Factors of aMTD Selected CPPs Newly Designed CPPs Critical Factor Range Range Bending Potential Proline presences in the Proline presences in the (Proline Position: PP) middle and/or at the middle (5′, 6′, 7′ or 8′) end of peptides and at the end of peptides Rigidity/Flexibility 45.5-57.3 (50.1 ± 3.6) 40-60 (Instability Index: II) Structural Feature 140.6-220.0 180-220 (Aliphatic Index: AI) (204.7 ± 37.5) Hydropathy  1.8-2.8 (2.4 ± 0.3) 2.1-2.6 (Grand Average of Hydropathy GRAVY) Length 11.6 ± 3.0  9-13 (Number of Amino Acid) Amino acid A, V, I, L, P A, V, I, L, P Composition

In Table 9, universal common features and sequence/structural motif are provided. Length is 9-13 amino acids, and bending potential is provided with the presence of proline in the middle of sequence (at 5′, 6′, 7′ or 8′ amino acid) for peptide bending and at the end of peptide for recombinant protein bending and Rigidity/Flexibility of aMTDs is II>40 are described in Table 9.

3-2. Critical Factors for Development of Advanced MTDs

Recombinant cell-permeable proteins fused to the hydrophobic CPPs to deliver therapeutically active cargo molecules including proteins into live cells had previously been reported, but the fusion proteins expressed in bacteria system were hard to be purified as a soluble form due to their low solubility and yield. To address the crucial weakness for further clinical development of the cell-permeable proteins as protein-based biotherapeutics, greatly improved form of the hydrophobic CPP, named as advanced MTD (aMTD) has newly been developed through critical factors-based peptide analysis. The critical factors used for the current invention of the aMTDs are herein (Table 9).

1. Amino Acid Length: 9-13

2. Bending Potential (Proline Position: PP)

: Proline presences in the middle (from 5′ to 8′ amino acid) and at the end of sequence

3. Rigidity/Flexibility (Instability Index: II): 40-60

4. Structural Feature (Aliphatic Index: AI): 180-220

5. Hydropathy (GRAVY): 2.1-2.6

6. Amino Acid Composition: Hydrophobic and Aliphatic amino acids—A, V, L, I and P

3-3. Design of Potentially Best aMTDs that all Critical Factors are Considered and Satisfied

After careful consideration of six critical factors derived from analysis of unique features of hydrophobic CPPs, advanced macromolecule transduction domains (aMTDs) have been designed and developed based on the common 12 amino acid platform which satisfies the critical factors including amino acid length (9-13) determined from the analysis.

Unlike previously published hydrophobic CPPs that require numerous experiments to determine their cell-permeability, newly developed aMTD sequences could be designed by performing just few steps as follows using above mentioned platform to follow the determined range value/feature of each critical factor.

First, prepare the 12 amino acid sequence platform for aMTD. Second, place proline (P) in the end (12′) of sequence and determine where to place proline in one of four U(s) in 5′, 6′, 7′, and 8. Third, alanine (A), valine (V), leucine (L) or isoleucine (I) is placed in either X(s) and/or U(s), where proline is not placed. Lastly, determine whether the amino acid sequences designed based on the platform, satisfy the value or feature of six critical factors to assure the cell permeable property of aMTD sequences. Through these processes, numerous novel aMTD sequences have been constructed. The expression vectors for preparing non-functional cargo recombinant proteins fused to each aMTD, expression vectors have been constructed and forcedly expressed in bacterial cells. These aMTD-fused recombinant proteins have been purified in soluble form and determined their cell-permeability quantitatively. aMTD sequences have been newly designed, numbered from 1 to 240, as shown in Table 10-15. In Table 10-15, sequence ID Number is a sequence listings for reference, and aMTD numbers refer to amino acid listing numbers that actually have been used at the experiments. For further experiments, aMTD numbers have been used. In addition, polynucleotide sequences shown in the sequence lists have been numbered from SEQ ID NO: 241 to SEQ ID NO: 480.

Tables 10 to 15 shows 240 new hydrophobic aMTD sequences that were developed to satisfy all critical factors.

TABLE 10 Sequence Rigidity/ Sturctural ID Flexibility Feature Hydropathy Residue Number aMTD Sequences Length (II) (AI) (GRAVY) structure  1  1 AAALAPVVLALP 12 57.3 187.6 2.1 Aliphatic  2  2 AAAVPLLAVVVP 12 41.3 195.0 2.4 Aliphatic  3  3 AALLVPAAVLAP 12 57.3 187.5 2.1 Aliphatic  4  4 ALALLPVAALAP 12 57.3 195.8 2.1 Aliphatic  5  5 AAALLPVALVAP 12 57.3 187.5 2.1 Aliphatic  6 11 VVALAPALAALP 12 57.3 187.5 2.1 Aliphatic  7 12 LLAAVPAVLLAP 12 57.3 211.7 2.3 Aliphatic  8 13 AAALVPVVALLP 12 57.3 203.3 2.3 Aliphatic  9 21 AVALLPALLAVP 12 57.3 211.7 2.3 Aliphatic 10 22 AVVLVPVLAAAP 12 57.3 195.0 2.4 Aliphatic 11 23 VVLVLPAAAAVP 12 57.3 195.0 2.4 Aliphatic 12 24 IALAAPALIVAP 12 50.2 195.8 2.2 Aliphatic 13 25 IVAVAPALVALP 12 50.2 203.3 2.4 Aliphatic 14 42 VAALPVVAVVAP 12 57.3 195.7 2.4 Aliphatic 15 43 LLAAPLVVAAVP 12 41.3 187.5 2.1 Aliphatic 16 44 ALAVPVALLVAP 12 57.3 203.3 2.3 Aliphatic 17 61 VAALPVLLAALP 12 57.3 211.7 2.3 Aliphatic 18 62 VALLAPVALAVP 12 57.3 203.3 2.3 Aliphatic 19 63 AALLVPALVAVP 12 57.3 203.3 2.3 Aliphatic

TABLE 11 Sequence Rigidity/ Sturctural ID Flexibility Feature Hydropathy Residue Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 20  64 AIVALPVAVLAP 12 50.2 203.3 2.4 Aliphatic 21  65 IAIVAPVVALAP 12 50.2 203.3 2.4 Aliphatic 22  81 AALLPALAALLP 12 57.3 204.2 2.1 Aliphatic 23  82 AVVLAPVAAVLP 12 57.3 195.0 2.4 Aliphatic 24  83 LAVAAPLALALP 12 41.3 195.8 2.1 Aliphatic 25  84 AAVAAPLLLALP 12 41.3 195.8 2.1 Aliphatic 26  85 LLVLPAAALAAP 12 57.3 195.2 2.1 Aliphatic 27 101 LVALAPVAAVLP 12 57.3 203.3 2.3 Aliphatic 28 102 LALAPAALALLP 12 57.3 204.2 2.1 Aliphatic 29 103 ALIAAPILALAP 12 57.3 204.2 2.2 Aliphatic 30 104 AVVAAPLVLALP 12 41.3 203.3 2.3 Aliphatic 31 105 LLALAPAALLAP 12 57.3 204.1 2.1 Aliphatic 32 121 AIVALPALALAP 12 50.2 195.2 2.2 Aliphatic 33 123 AAIIVPAALLAP 12 50.2 195.2 2.2 Aliphatic 34 124 IAVALPALIAAP 12 50.3 195.2 2.2 Aliphatic 35 141 AVIVLPALAVAP 12 50.2 203.3 2.4 Aliphatic 36 143 AVLAVPAVLVAP 12 57.3 195.0 2.4 Aliphatic 37 144 VLAIVPAVALAP 12 50.2 203.3 2.4 Aliphatic 38 145 LLAVVPAVALAP 12 57.3 203.3 2.3 Aliphatic 39 161 AVIALPALIAAP 12 57.3 195.2 2.2 Aliphatic 40 162 AVVALPAALIVP 12 50.2 203.3 2.4 Aliphatic 41 163 LALVLPAALAAP 12 57.3 195.8 2.1 Aliphatic 42 164 LAAVLPALLAAP 12 57.3 195.8 2.1 Aliphatic 43 165 ALAVPVALAIVP 12 50.2 203.3 2.4 Aliphatic 44 182 ALIAPVVALVAP 12 57.3 203.3 2.4 Aliphatic 45 183 LLAAPVVIALAP 12 57.3 211.6 2.4 Aliphatic 46 184 LAAIVPAIIAVP 12 50.2 211.6 2.4 Aliphatic 47 185 AALVLPLIIAAP 12 41.3 220.0 2.4 Aliphatic 48 201 LALAVPALAALP 12 57.3 195.8 2.1 Aliphatic 49 204 LIAALPAVAALP 12 57.3 195.2 2.2 Aliphatic 50 205 ALALVPAIAALP 12 57.3 195.2 2.2 Aliphatic 51 221 AAILAPIVALAP 12 50.2 195.2 2.2 Aliphatic 52 222 ALLIAPAAVIAP 12 57.3 195.2 2.2 Aliphatic 53 223 AILAVPIAVVAP 12 57.3 203.3 2.4 Aliphatic 54 224 ILAAVPIALAAP 12 57.3 195.2 2.2 Aliphatic 55 225 VAALLPAAAVLP 12 57.3 187.5 2.1 Aliphatic 56 241 AAAVVPVLLVAP 12 57.3 195.0 2.4 Aliphatic 57 242 AALLVPALVAAP 12 57.3 187.5 2.1 Aliphatic 58 243 AAVLLPVALAAP 12 57.3 187.5 2.1 Aliphatic 59 245 AAALAPVLALVP 12 57.3 187.5 2.1 Aliphatic 60 261 LVLVPLLAAAAP 12 41.3 211.6 2.3 Aliphatic 61 262 ALIAVPAIIVAP 12 50.2 211.6 2.4 Aliphatic 62 263 ALAVIPAAAILP 12 54.9 195.2 2.2 Aliphatic 63 264 LAAAPVVIVIAP 12 50.2 203.3 2.4 Aliphatic 64 265 VLAIAPLLAAVP 12 41.3 211.6 2.3 Aliphatic 65 281 ALIVLPAAVAVP 12 50.2 203.3 2.4 Aliphatic 66 282 VLAVAPALIVAP 12 50.2 203.3 2.4 Aliphatic 67 283 AALLAPALIVAP 12 50.2 195.8 2.2 Aliphatic 68 284 ALIAPAVALIVP 12 50.2 211.7 2.4 Aliphatic 69 285 AIVLLPAAVVAP 12 50.2 203.3 2.4 Aliphatic

TABLE 12 Sequence Rigidity/ Sturctural ID Flexibility Feature Hydropathy Residue Number aMTD Sequences Length (II) (AI) (GRAVY) Structure  70 301 VIAAPVLAVLAP 12 57.3 203.3 2.4 Aliphatic  71 302 LALAPALALLAP 12 57.3 204.2 2.1 Aliphatic  72 304 AIILAPIAAIAP 12 57.3 204.2 2.3 Aliphatic  73 305 IALAAPILLAAP 12 57.3 204.2 2.2 Aliphatic  74 321 IVAVALPALAVP 12 60.2 203.3 2.3 Aliphatic  75 322 VVAIVLPALAAP 12 50.2 203.3 2.3 Aliphatic  76 323 IVAVALPVALAP 12 50.2 203.3 2.3 Aliphatic  77 324 IVAVALPAALVP 12 50.2 203.3 2.3 Aliphatic  78 325 IVAVALPAVALP 12 50.2 203.3 2.3 Aliphatic  79 341 IVAVALPAVLAP 12 50.2 203.3 2.3 Aliphatic  80 342 VIVALAPAVLAP 12 50.2 203.3 2.3 Aliphatic  81 343 IVAVALPALVAP 12 50.2 203.3 2.3 Aliphatic  82 345 ALLIVAPVAVAP 12 50.2 203.3 2.3 Aliphatic  83 361 AVVIVAPAVIAP 12 50.2 195.0 2.4 Aliphatic  84 363 AVLAVAPALIVP 12 60.2 203.3 2.3 Aliphatic  85 364 LVAAVAPALIVP 12 50.2 203.3 2.3 Aliphatic  86 365 AVIVVAPALLAP 12 50.3 203.3 2.3 Aliphatic  87 381 VVAIVLPAVAAP 12 50.2 195.0 2.4 Aliphatic  88 382 AAALVIPAILAP 12 54.9 195.8 2.2 Aliphatic  89 383 VIVALAPALLAP 12 50.2 211.6 2.3 Aliphatic  90 384 VIVAIAPALLAP 12 50.2 211.6 2.4 Aliphatic  91 385 IVAIAVPALVAP 12 50.2 203.3 2.4 Aliphatic  92 401 AALAVIPAAILP 12 54.9 195.8 2.2 Aliphatic  93 402 ALAAVIPAAILP 12 54.9 195.3 2.2 Aliphatic  94 403 AAALVIPAAILP 12 54.9 195.8 2.2 Aliphatic  95 404 LAAAVIPAAILP 12 54.9 195.8 2.2 Aliphatic  96 405 LAAAVIPVAILP 12 54.9 211.7 2.4 Aliphatic  97 421 AAILAAPLIAVP 12 57.3 195.8 2.2 Aliphatic  98 422 VVAILAPLLAAP 12 57.3 211.7 2.4 Aliphatic  99 424 AVVVAAPVLALP 12 57.3 195.0 2.4 Aliphatic 100 425 AVVAIAPVLALP 12 67.3 203.3 2.4 Aliphatic 101 442 ALAALVPAVLVP 12 57.3 203.3 2.2 Aliphatic 102 443 ALAALVPVALVP 12 57.3 203.3 2.3 Aliphatic 103 444 LAAALVPVALVP 12 57.3 203.3 2.3 Aliphatic 104 446 ALAALVPALVVP 12 57.3 203.3 2.3 Aliphatic 105 461 IAAVIVPAVALP 12 50.2 203.3 2.4 Aliphatic 106 462 IAAVLVPAVALP 12 57.3 203.3 2.4 Aliphatic 107 463 AVAILVPLLAAP 12 57.3 211.7 2.4 Aliphatic 108 464 AVVILVPLAAAP 12 57,3 203.3 2.4 Aliphatic 109 465 IAAVIVPVAALP 12 50.2 203.3 2.4 Aliphatic 110 481 AlAIAIVPVALP 12 50.2 211.6 2.4 Aliphatic 111 482 ILAVAAIPVAVP 12 54.9 203.3 2 4 Aliphatic 112 483 ILAAAIIPAALP 12 54.9 204.1 2 2 Aliphatic 113 484 LAVVLAAPAIVP 12 50.2 203.3 2 4 Aliphatic 114 485 AILAAIVPLAVP 12 50.2 211.6 2.4 Aliphatic 115 501 VIVALAVPALAP 12 50.2 203.3 2 4 Aliphatic 116 502 AIVALAVPVLAP 12 50.2 203.3 2.4 Aliphatic 117 503 AAIIIVLPAALP 12 50.2 220.0 2 4 Aliphatic 118 504 LIVALAVPALAP 12 50.2 211.7 2 4 Aliphatic 119 505 AIIIVIAPAAAP 12 50.2 195.8 2.3 Aliphatic

TABLE 13 Sequence Rigidity/ Sturctural ID Flexibility Feature Hydropathy Residue Number aMTD Sequences Length (II) (AI) (GRAVY) structure 120 521 LAALIVVPAVAP 12 50.2 203.3 2.4 Aliphatic 121 522 ALLVIAVPAVAP 12 57.3 203.3 2.4 Aliphatic 122 524 AVALIVVPALAP 12 50.2 203.2 2.4 Aliphatic 123 525 ALAIVVAPVAVP 12 50.2 195.0 2.4 Aliphatic 124 541 LLALIIAPAAAP 12 57.3 204.1 2.1 Aliphatic 125 542 ALALIIVPAVAP 12 50.2 211.6 2.4 Aliphatic 126 543 LLAALIAPAALP 12 57.3 204.1 2.1 Aliphatic 127 544 IVALIVAPAAVP 12 43.1 203.3 2.4 Aliphatic 128 545 VVLVLAAPAAVP 12 57.3 195.0 2.3 Aliphatic 129 561 AAVAIVLPAVVP 12 50.2 195.0 2.4 Aliphatic 130 562 ALIAAIVPALVP 12 50.2 211.7 2.4 Aliphatic 131 563 ALAVIVVPALAP 12 50.2 203.3 2.4 Aliphatic 132 564 VAIALIVPALAP 12 50.2 211.7 2.4 Aliphatic 133 565 VAIVLVAPAVAP 12 50.2 195.0 2.4 Aliphatic 134 582 VAVALIVPALAP 12 50.2 203.3 2.4 Aliphatic 135 583 AVILALAPIVAP 12 50.2 211.6 2.4 Aliphatic 136 585 ALIVAIAPALVP 12 50.2 211.6 2.4 Aliphatic 137 601 AAILIAVPIAAP 12 57.3 195.8 2.3 Aliphatic 138 602 VIVALAAPVLAP 12 50.2 203.3 2.4 Aliphatic 139 603 VLVALAAPVIAP 12 57.3 203.3 2.4 Aliphatic 140 604 VALIAVAPAVVP 12 57.3 195.0 2.4 Aliphatic 141 605 VIAAVLAPVAVP 12 57.3 195.0 2.4 Aliphatic 142 622 ALIVLAAPVAVP 12 50.2 203.3 2.4 Aliphatic 143 623 VAAAIALPAIVP 12 50.2 187.5 2.3 Aliphatic 144 625 ILAAAAAPLIVP 12 50.2 195.8 2.2 Aliphatic 145 643 LALVLAAPAIVP 12 50.2 211.6 2.4 Aliphatic 146 645 ALAVVALPAIVP 12 50.2 203.3 2.4 Aliphatic 147 661 AAILAPIVAALP 12 50.2 195.8 2.2 Aliphatic 148 664 ILIAIAIPAAAP 12 54.9 204.1 2.3 Aliphatic 149 665 LAIVLAAPVAVP 12 50,2 203.3 2.3 Aliphatic 150 666 AAIAIIAPAIVP 12 50.2 195.8 2.3 Aliphatic 151 667 LAVAIVAPALVP 12 60.2 203.3 2.3 Aliphatic 152 683 LAIVLAAPAVLP 12 50.2 211.7 2.4 Aliphatic 153 684 AAIVLALPAVLP 12 50.2 211.7 2.4 Aliphatic 154 685 ALLVAVLPAALP 12 57.3 211.7 2.3 Aliphatic 155 686 AALVAVLPVALP 12 57.3 203.3 2.3 Aliphatic 156 687 AILAVALPLLAP 12 57.3 220.0 2.3 Aliphatic 157 703 IVAVALVPALAP 12 50.2 203.3 2.4 Aliphatic 158 705 IVAVALLPALAP 12 50.2 211.7 2.4 Aliphatic 159 706 IVAVALLPAVAP 12 50.2 203.3 2.4 Aliphatic 160 707 IVALAVLPAVAP 12 50.2 203.3 2.4 Aliphatic 161 724 VAVLAVLPALAP 12 57.3 203.2 2.3 Aliphatic 162 725 IAVLAVAPAVLP 12 57.3 203.3 2.3 Aliphatic 163 726 LAVAIIAPAVAP 12 57.3 187.5 2.2 Aliphatic 164 727 VALAIALPAVLP 12 57.3 211.6 2.3 Aliphatic 165 743 AIAIALVPVALP 12 57.3 211.6 2.4 Aliphatic 166 744 AAVVIVAPVALP 12 50.2 195.0 2.4 Aliphatic 167 746 VAIIVVAPALAP 12 50.2 203.3 2.4 Aliphatic 168 747 VALLALAPALAP 12 57.3 195.8 2.2 Aliphatic 169 763 VAVLIAVPALAP 12 57.3 203.3 2.3 Aliphatic

TABLE 14 Sequence Rigidity/ Sturctural ID Flexibility Feature Hydropathy Residue Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 170 764 AVALAVLPAVVP 12 57.3 195.0 2.3 Aliphatic 171 765 AVALAVVPAVLP 12 57.3 195.0 2.3 Aliphatic 172 766 IVVIAVAPAVAP 12 50.2 195.0 2.4 Aliphatic 173 767 IVVAAVVPALAP 12 50.2 195.0 2.4 Aliphatic 174 783 IVALVPAVAIAP 12 50.2 203.3 2.5 Aliphatic 175 784 VAALPAVALVVP 12 57.3 195.0 2.4 Aliphatic 176 786 LVAIAPLAVLAP 12 41.3 211.7 2.4 Aliphatic 177 787 AVALVPVIVAAP 12 50.2 195.0 2.4 Aliphatic 178 788 AIAVAIAPVALP 12 57.3 187.5 2.3 Aliphatic 179 803 AIALAVPVLALP 12 57.3 211.7 2.4 Aliphatic 180 805 LVLIAAAPIALP 12 41.3 220.0 2.4 Aliphatic 181 806 LVALAVPAAVLP 12 57.3 203.3 2.3 Aliphatic 182 807 AVALAVPALVLP 12 57.3 203.3 2.3 Aliphatic 183 808 LVVLAAAPLAVP 12 41.3 203.3 2.3 Aliphatic 184 809 LIVLAAPALAAP 12 50.2 195.8 2.2 Aliphatic 185 810 VIVLAAPALAAP 12 50.2 187.5 2.2 Aliphatic 186 811 AVVLAVPALAVP 12 57.3 195.0 2.3 Aliphatic 187 824 LIIVAAAPAVAP 12 50.2 187.5 2.3 Aliphatic 188 825 IVAVIVAPAVAP 12 43.2 195.0 2.5 Aliphatic 189 826 LVALAAPIIAVP 12 41.3 211.7 2.4 Aliphatic 190 827 IAAVLAAPALVP 12 57.3 187.5 2.2 Aliphatic 191 828 IALLAAPIIAVP 12 41.3 220.0 2.4 Aliphatic 192 829 AALALVAPVIVP 12 50.2 203.3 2.4 Aliphatic 193 830 IALVAAPVALVP 12 57.3 203.3 2.4 Aliphatic 194 831 IIVAVAPAAIVP 12 43.2 203.3 2.5 Aliphatic 195 832 AVAAIVPVIVAP 12 43.2 195.0 2.5 Aliphatic 196 843 AVLVLVAPAAAP 12 41.3 219.2 2.5 Aliphatic 197 844 VVALLAPLIAAP 12 41.3 211.8 2.4 Aliphatic 198 845 AAVVIAPLLAVP 12 41.3 203.3 2.4 Aliphatic 199 846 IAVAVAAPLLVP 12 41.3 203.3 2.4 Aliphatic 200 847 LVAIVVLPAVAP 12 50.2 219.2 2.6 Aliphatic 201 848 AVAIVVLPAVAP 12 50.2 195.0 2.4 Aliphatic 202 849 AVILLAPLIAAP 12 57.3 220.0 2.4 Aliphatic 203 850 LVIALAAPVALP 12 57.3 211.7 2.4 Aliphatic 204 851 VLAVVLPAVALP 12 57.3 219.2 2.5 Aliphatic 205 852 VLAVAAPAVLLP 12 57.3 203.3 2.3 Aliphatic 206 863 AAVVLLPIIAAP 12 41.3 211.7 2.4 Aliphatic 207 864 ALLVIAPAIAVP 12 57.3 211.7 2.4 Aliphatic 208 865 AVLVIAVPAIAP 12 57.3 203.3 2.5 Aliphatic 209 867 ALLVVIAPLAAP 12 41.3 211.7 2.4 Aliphatic 210 868 VLVAAILPAAIP 12 64.2 211.7 2.4 Aliphatic 211 870 VLVAAVLPIAAP 12 41.3 203.3 2.4 Aliphatic 212 872 VLAAAVLPLVVP 12 41.3 219.2 2.5 Aliphatic 213 875 AIAIVVPAVAVP 12 50.2 195.0 2.4 Aliphatic 214 877 VAILAVPAVVAP 12 57.3 195.0 2.4 Aliphatic 215 878 IVALVAPAAVVP 12 50.2 195.0 2.4 Aliphatic 216 879 AAIVLLPAVVVP 12 50.2 219.1 2.5 Aliphatic 217 881 AALIVVPAVAVP 12 50.2 195.0 2.4 Aliphatic 218 882 AIALVVPAVAVP 12 57.3 195.0 2.4 Aliphatic 219 883 LAIVPAAIAALP 12 50.2 195.8 2.2 Aliphatic

TABLE 15 Sequence Rigidity/ Sturctural ID Flexibility Feature Hydropathy Residue Number aMTD Sequences Length (II) (AI) (GRAVY) Structure 220 885 LVAIAPAVAVLP 12 57.3 203.3 2.4 Aliphatic 221 887 VLAVAPAVAVLP 12 57.3 195.0 2.4 Aliphatic 222 888 ILAVVAIPAAAP 12 54.9 187.6 2.3 Aliphatic 223 889 ILVAAAPIAALP 12 57.3 195.8 2.2 Aliphatic 224 891 ILAVAAIPAALP 12 54.9 195.8 2.2 Aliphatic 225 893 VIAIPAILAAAP 12 54.9 195.8 2.3 Aliphatic 226 895 AIIIVVPAIAAP 12 50.2 211.7 2.5 Aliphatic 227 896 AILIVVAPLAAP 12 50.2 211.7 2.5 Aliphatic 228 897 AVIVPVAIIAAP 12 50.2 203.3 2.5 Aliphatic 229 899 AVVIALPAVVAP 12 57.3 195.0 2.4 Aliphatic 230 900 ALVAVIAPVVAP 12 57.3 195.0 2.4 Aliphatic 231 901 ALVAVLPAVAVP 12 67.3 195.0 2.4 Aliphatic 232 902 ALVAPLLAVAVP 12 41.3 203.3 2.3 Aliphatic 233 904 AVLAVVAPVVAP 12 57.3 186.7 2.4 Aliphatic 234 905 AVIAVAPLVVAP 12 41.3 195.0 2.4 Aliphatic 235 906 AVIALAPVVVAP 12 57.3 195.0 2.4 Aliphatic 236 907 VAIALAPVVVAP 12 67.3 195.0 2.4 Aliphatic 237 908 VALALAPVVVAP 12 57.3 195.0 2.3 Aliphatic 238 910 VAALLPAVVVAP 12 57.3 195.0 2.3 Aliphatic 239 911 VALALPAVVVAP 12 57.3 195.0 2.3 Aliphatic 240 912 VALLAPAVVVAP 12 57.3 195.0 2.3 Aliphatic 52.6 ± 5.1 201.7 ± 7.8 2.3 ± 0.1

3-4. Design of the Peptides that Did not Satisfy at Least One Critical Factor

To demonstrate that this invention of new hydrophobic CPPs-aMTDs, which satisfy all critical factors described above, are correct and rationally designed, the peptides which do not satisfy at least one critical factor have also been designed. Total of 31 rPeptides (rPs) are designed, developed and categorized as follows: no bending peptides, either no proline in the middle as well at the end and/or no central proline; rigid peptides (II<40); too much flexible peptides; aromatic peptides (aromatic ring presences); hydrophobic, with non-aromatic peptides but have amino acids other than A, V, L, I, P or additional proline residues; hydrophilic, but non-aliphatic peptides.

3-4-1. Peptides that do not Satisfy the Bending Potential

Table 16 shows the peptides that do not have any proline in the middle (at 5′, 6′, 7′ or 8′) and at the end of the sequences. In addition, Table 16 describes the peptides that do not have proline in the middle of the sequences. All these peptides are supposed to have no-bending potential.

TABLE 16 Proline Rigidity/ Sturctural rPeptide Position Flexibility Feature Hydropathy Group ID Sequences Length (PP) (II) (AI) (GRAVY) No-Bending 931 AVLIAPAILAAA 12  6 57.3 204.2  2.5 Peptides 936 ALLILAAAVAAP 12 12 41.3 204.2  2.4 (No Proline 152 LAAAVAAVAALL 12 None  9.2 204.2  2.7 at 5, 6, 7  27 LAIVAAAAALVA 12 None  2.1 204.2  2.8 or 8 and/ 935 ALLILPAAAVAA 12  6 57.3 204.2  2.4 or 12) 670 ALLILAAAVAAL 12 None 25.2 236.6  2.8 934 LILAPAAVVAAA 12  5 57.3 195.8  2.5  37 TTCSQQQVCTNG 12 None 53.1   0.0 -1.1  16 NNSCTTYTNGSQ 12 None 47.4   0.0 -1.4 113 PVAVALLIAVPP 12 1,11,12 57.3 195.0  2.1

3-4-2. Peptides that do not Satisfy the Rigidity/Flexibility

To prove that rigidity/flexibility of the sequence is a crucial critical factor, rigid (Avg. II: 21.8±6.6) and too high flexible sequences (Avg. II: 82.3±21.0) were also designed. Rigid peptides that instability index is much lower than that of new aMTDs (II: 41.3-57.3, Avg. II: 53.3±5.7) are shown in Table 17. Bending, but too high flexible peptides that II is much higher than that of new aMTDs are also provided in Table 18.

TABLE 17 Proline Rigidity/ Sturctural rPeptide Position Flexibility Feature Hydropathy Group ID Sequences Length (PP) (II) (AI) (GRAVY) Rigid 226 ALVAAIPALAIP 12 6    20.4  195.8  2.2 Peptides   6 VIAMIPAAFWVA 12 6    15.7  146.7  2.2 (II < 50) 750 LAIAAIAPLAIP 12 8.12 22.8  204.2  2.2  26 AAIALAAPLAIV 12 8    18.1  204.2  2.5 527 LVIAAVAPIAIP 12 8.12 22.9  211.7  2.4 466 IIAAAAPLAIIP 12 7.12 22.6  204.2  2.3 167 VAJAIPAALAIP 12 6.12 20.4  195.8  2.3 246 VVAVPLLVAFAA 12 5    25.2  195.0  2.7 426 AAALAIPLAIIP 12 7.12  4.37 204.2  2.2 606 AAAIAAIPIIIP 12 8.12  4.4  204.2  2.4  66 AGVLGGPIMGVP 12 7.12 35.5  121.7  1.3 246 VAAIVPIAALVP 12 6.12 34.2  203.3  2.5 227 LAAIVPIAAAVP 12 6.12 24.2  187.5  2.2  17 GGCSAPQTTCSN 12 6    51.6    6.3 -0.5  67 LDAEVPLADDVP 12 6.12 34.2  130.0  0.3

TABLE 18 Proline Rigidity/ Sturctural rPeptide Position Flexibility Feature Hydropathy Group ID Sequences Length (PP) (II) (AI) (GARVY) Bending 692 PAPLPPVVILAV 12 1,3,5,6 105.5 186.7 1.8 Peptides  69 PVAVLPPAALVP 12 1,6,7,12  89.4 162.5 1.6 but Too 390 VPLLVPVVPVVP 12 2,6,9,12 105.4 210.0 2.2 High 350 VPILVPVVPVVP 12 2,6,9,12 121.5 210.0 2.2 Flexibility 331 VPVLVPLVPVVP 12 2,6,9,12 105.4 210.0 2.2   9 VALVPAALILPP 12 5,11,12  89.4 203.3 2.1  68 VAPVLPAAPLVP 12 3,6,9,12 105.5 162.5 1.6 349 VPVLVPVVPVVP 12 2,6,9,12 121.5 201.6 2.2 937 VPVLVPLPVPVV 12 2,6,8,10 121.5 210.0 2.2 938 VPVLLPVVVPVP 12 2,6,10,12 121.5 210.0 2.2 329 LPVLVPVVPVVP 12 2,6,9,12 121.5 210.0 2.2  49 VVPAAPAVPVVP 12 3,6,9,12 121.5 145.1 1.3 772 LIVAPVIPIIVP 12 2,5,8,12  79.9 210.8 2.1 210 ALIALPALPALP 12 6,9,12  89.4 195.8 1.8  28 AVPLLPLVPAVP 12 3,6,9,12  89.4 186.1 1.8 693 AAPVLPVAVPIV 12 3,6,10  82.3 186.7 2.1 169 VALVAPALILAP 12 6,12  73.4 211.7 2.4  29 VLPPLPVLPVLP 12 3,4,6, 121.5 202.5 1.7 9,12 190 AAILAPAVIAPP 12 6,11,12  89.4 163.3 1.8

3-4-3. Peptides that do not satisfy the structural features

New hydrophobic CPPs-aMTDs are consisted with only hydrophobic and aliphatic amino acids (A, V, L, I and P) with average ranges of the indexes—AI: 180-220 and GRAVY: 2.1-2.6 (Table 9). Based on the structural indexes, the peptides which contain an aromatic residue (W, F or Y) are shown in Table 19 and the peptides which are hydrophobic with non-aromatic sequences but have amino acids residue other than A, V, L, I, P or additional proline residues are designed (Table 20). Finally, hydrophilic and/or bending peptides which are consisted with non-aliphatic amino acids are shown in Table 21.

TABLE 19 Proline Rigidity/ Sturctural rPeptide Position Flexibility Feature Hydropathy Group ID Sequences Length (PP) (II) (AI) (GRAVY) Aromatic  30 WFFAGPIMLIWP 12 6,12   9.2 105.9 1.4 Peptides  33 AAAILAPAFLAV 12 7  57.3 171.7 2.4 (Aromatic 131 WIIAPVWLAWIA 12 5  51.6 179.2 1.9 Ring 922 WYVIFV1PLVVP 12 9,12  41.3 194.2 2.2 Presences)  71 FMWMWFPFMWYP 12 7,12 713     0.0 0.6 921 IWWFVVLPLVVP 12 8,12 413   194.2 2.2

TABLE 20 Proline Rigidity/ Sturctural rPeptide Position Flexibility Feature Hydropathy Group ID Sequences Length (PP) (II) (AI) (GARVY) Hydrophobic 436 VVMLVVPAVMLP 12 7,12 57.3 194.2 2.6 but Non 138 PPAALLAILAVA 12 1,2 57.3 195.2 2.2 Aromatic  77 PVALVLVALVAP 12 1,12 41.3 219.2 2.5 Peptides 577 MLMIALVPMIAV 12 8 18.9 195.0 2.7  97 ALLAAPPALLAL 12 6,7 57.3 204.2 2.1 214 ALIVAPALMALP 12 6,12 60.5 187.5 2.2  59 AVLAAPVVAALA 12 6 41.3 187.5 2.5  54 LAVAAPPVVALL 12 6,7 57.3 203.3 2.3

TABLE 21 Proline Rigidity/ Structural rPeptide Position Flexibility Feature Hydropathy Group ID Sequences Length (PP) (II) (AI) (GRAVY) Hydrophilic 949 SGNSCQQCGNSS 12 None 41.7 0.0 -1.1 Peptides  39 CYNTSPCTGCCY 12 6 52.5 0.0  0.0 but Non  19 YVSCCTYTNGSQ 12 None 47.7 0.0 -1.0 Aliphatic 947 CYYNQQSNNNNQ 12 None 59.6 0.0 -2.4 139 TGSTNSPTCTST 12 7 53.4 0.0 -0.7  18 NYCCIPTINGQS 12 6 47.9 0.0 -0.9  20 NYCNTCPTYGQS 12 7 47.4 0.0 -0.9 635 GSTGGSQQNNQY 12 None 31.9 0.0 -1.9  40 TYNTSCTPGTCY 12 8 49.4 0.0 -0.6  57 QNNCNTSSQGGG 12 None 52.4 0.0 -1.6 159 CYSGSTSQNQPP 12 11,12 51.0 0.0 -1.3 700 GTSNTCQSNQNS 12 None 19.1 0.0 -1.6  38 YYNQSTCGGQCY 12 None 53.8 0.0 -1.0

3-5. Summary of Newly Designed Peptides

Total of 457 sequences have been designed based on the critical factors. Designed potentially best aMTDs (hydrophobic, flexible, bending, aliphatic and 12-A/a length peptides) that do satisfy all range/feature of critical factors are 316. Designed rPeptides that do not satisfy at least one of the critical factors are 141 that no bending peptide sequences are 26; rigid peptide (II<40) sequences are 23; too much flexible peptides are 24; aromatic peptides (aromatic ring presences) are 27; hydrophobic, but non-aromatic peptides are 23; and hydrophilic, but non-aliphatic peptides are 18.

4. Preparation of Recombinant Report Proteins Fused to aMTDs and rPeptides

Recombinant proteins fused to aMTDs and others [rPeptides, reference hydrophobic CPP sequences (MTM and MTD)] were expressed in a bacterial system, purified with single-step affinity chromatography and prepared as soluble proteins in physiological condition. These recombinant proteins have been tested for the ability of their cell-permeability by utilizing flow cytometry and laser scanning confocal microscopy.

4-1. Selection of Cargo Protein for Recombinant Proteins Fused to Peptide Sequences

For clinical/non-clinical application, aMTD-fused cargo materials would be biologically active molecules that could be one of the following: enzymes, transcription factors, toxic, antigenic peptides, antibodies and antibody fragments. Furthermore, biologically active molecules could be one of these following macromolecules: enzymes, hormones, carriers, immunoglobulin, membrane-bound proteins, transmembrane proteins, internal proteins, external proteins, secreted proteins, virus proteins, native proteins, glycoproteins, fragmented proteins, disulfide bonded proteins, recombinant proteins, chemically modified proteins and prions. In addition, these biologically active molecules could be one of the following: nucleic acid, coding nucleic acid sequence, mRNAs, antisense RNA molecule, carbohydrate, lipid and glycolipid.

According to these pre-required conditions, a non-functional cargo to evaluate aMTD-mediated protein uptake has been selected and called as Cargo A (CRA) that should be soluble and non-functional. The domain (A/a 289-840; 184 A/a length) is derived from protein S (Genbank ID: CP000113.1).

4-2. Construction of Expression Vector and Preparation of Recombinant Proteins

Coding sequences for recombinant proteins fused to each aMTD are cloned Nde1 (5′) and SalI (3′) in pET-28a(+) (Novagen, Darmstadt, Germany) from PCR-amplified DNA segments. PCR primers for the recombinant proteins fused to aMTD and rPeptides are SEQ ID NOs: 481˜797. Structure of the recombinant proteins is displayed in FIG. 1.

The recombinant proteins were forcedly expressed in E. coli BL21 (DE3) cells grown to an OD₆₀₀ of 0.6 and induced for 2 hours with 0.7 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The proteins were purified by Ni²⁺ affinity chromatography as directed by the supplier (Qiagen, Hilden, Germany) in natural condition. After the purification, purified proteins were dissolved in a physiological buffer such as DMEM medium.

TABLE 22 Potentially Best aMTDs (Hydrophobic, Flexible, 240  Bending, Aliphatic & Helical) Random Peptides 31 No Bending Peptides (No Proline at 5 or 6 and/or 12) 02 No Bending Peptides (No Central Proline) 01 Rigid Peptides (II < 50) 09 Too Much Flexible Peptides 09 Aromatic Peptides (Aromatic Ring Presences) 01 Hydrophobic, But Non-Aromatic Peptides 02 Hydrophilic, But Non-Aliphatic Peptides 07

4-3. Expression of aMTD- or Random Peptide (rP)-Fused Recombinant Proteins

Using the standardized six critical factors, 316 aMTD sequences have been designed. In addition, 141 rPeptides are also developed that lack one of these critical factors: no bending peptides: i) absence of proline both in the middle and at the end of sequence or ii) absence of proline either in the middle or at the end of sequence, rigid peptides, too much flexible peptides, aromatic peptides (aromatic ring presence), hydrophobic but non-aromatic peptides, and hydrophilic but non-aliphatic peptides (Table 22).

These rPeptides are devised to be compared and contrasted with aMTDs in order to analyze structure/sequence activity relationship (SAR) of each critical factor with regard to the peptides' intracellular delivery potential. All peptide (aMTD or rPeptide)-containing recombinant proteins have been fused to the CRA to enhance the solubility of the recombinant proteins to be expressed, purified, prepared and analyzed.

These designed 316 aMTDs and 141 rPeptides fused to CRA were all cloned (FIG. 2) and tested for inducible expression in E. coli (FIG. 3). Out of these peptides, 240 aMTDs were inducibly expressed, purified and prepared in soluble form (FIG. 4). In addition, 31 rPeptides were also prepared as soluble form (FIG. 4).

To prepare the proteins fused to rPeptides, 60 proteins were expressed that were 10 out of 26 rPeptides in the category of no bending peptides (Table 16); 15 out of 23 in the category of rigid peptides [instability index (II)<40] (Table 17); 19 out of 24 in the category of too much flexible peptides (Table 18); 6 out of 27 in the category of aromatic peptides (Table 19); 8 out of 23 in the category of hydrophobic but non-aromatic peptides (Table 20); and 12 out of 18 in the category of hydrophilic but non-aliphatic peptides (Table 21).

4-4. Quantitative Cell-Permeability of aMTD-Fused Recombinant Proteins

The aMTDs and rPeptides were fluorescently labeled and compared based on the critical factors for cell-permeability by using flow cytometry and confocal laser scanning microscopy (FIGS. 5 to 8). The cellular uptake of the peptide-fused non-functional cargo recombinant proteins could quantitatively be evaluated in flow cytometry, while confocal laser scanning microscopy allows intracellular uptake to be assessed visually. The analysis included recombinant proteins fused to a negative control [rP38] that has opposite characteristics (hydrophilic and aromatic sequence: YYNQSTCGGQCY) to the aMTDs (hydrophobic and aliphatic sequences). Relative cell-permeability (relative fold) of aMTDs to the negative control was also analyzed (Table 23 and FIG. 9).

Table 23 shows Comparison Analysis of Cell-Permeability of aMTDs with a Negative Control (A: rP38).

TABLE 23 Negative Control rP38 aMTD 19.6 ± 1.6* The Average of 240 aMTDs (Best: 164.2) *Relative Fold (aMTD in Geo Mean in its comparison to rP38)

Relative cell-permeability (relative fold) of aMTDs to the reference CPPs [B: MTM12 (AAVLLPVLLAAP), C: MTD85 (AVALLILAV)] was also analyzed (Tables 40 and 41)

Table 24 shows Comparison Analysis of Cell-Permeability of aMTDs with a Reference CPP (B: MTM12).

TABLE 24 MTM12 aMTD 13.1 ± 1.1* The Average of 240 aMTDs (Best: 109.9) *Relative Fold (aMTD in Geo Mean in its comparison to MTM12)

Table 25 shows Comparison Analysis of Cell-Permeability of aMTDs with a Reference CPP (C: MTD85).

TABLE 25 MTD85 aMTD 6.6 ± 0.5* The Average of 240 aMTDs (Best: 55.5) *Relative Fold (aMTD in Geo Mean in its comparison to MTD85)

Geometric means of negative control (histidine-tagged rP38-fused CRA recombinant protein) subtracted by that of naked protein (histidine-tagged CRA protein) lacking any peptide (rP38 or aMTD) was standardized as relative fold of 1. Relative cell-permeability of 240 aMTDs to the negative control (A type) was significantly increased by up to 164 fold, with average increase of 19.6±1.6 (Table 26-31).

TABLE 26 Sequence Proline Rigidity/ Sturctural Relative ID Position Flexibility Feature Hydropathy Ratio (Fold) Number aMTD Sequences Length (PP) (II) (AI) (GRAVY) A B C  1 898 AVVIALPAVVAP 12 7 57.3 195.0 2.4 164.2 109.9 55.5  2 908 VALALAPVVVAP 12 7 57.3 195.0 2.3 150.6 100.8 50.9  3 910 VAALLPAVVVAP 12 6 57.3 195.0 2.3 148.5  99.4 50.2  4 810 VIVLAAPALAAP 12 7 50.2 187.5 2.2 120.0  80.3 40.6  5 904 AVLAVVAPVVAP 12 8 57.3 186.7 2.4 105.7  70.8 35.8  6 321 IVAVALPALAVP 12 7 50.2 203.3 2.3  97.8  65.2 32.9  7 851 VLAVVLPAVALP 12 7 57.3 219.2 2.5  96.6  64.7 32.7  8 911 VALALPAVVVAP 12 6 57.3 195.0 2.3  84.8  56.8 28.7  9 852 VLAVAAPAVLLP 12 7 57.3 203.3 2.3  84.6  56.6 28.6 10 803 AIALAVPVLALP 12 7 57.3 211.7 2.4  74.7  50.0 25.3 11 888 ILAVVAIPAAAP 12 8 54.9 187.5 2.2  71.0  47.5 24.0 12 825 IVAVIVAPAVAP 12 8 43.2 196.0 2.5  69.7  46.6 23.6 13 895 AIIIVVPAIAAP 12 7 50.2 211.7 2.5  60.8  40.7 20.6 14 896 AILIVVAPIAAP 12 8 50.2 211.7 2.5  57.5  38.5 19.4 15 727 VALAIALPAVLP 12 8 57.3 211.6 2.3  54.7  36.7 18.5 16 603 VLVALAAPVIAP 12 8 57.3 203.3 2.4  54.1  36.1 18.2 17 847 LVAIVVLPAVAP 12 8 50.2 219.2 2.6  50.2  33.4 16.9 18 826 LVALAAPIIAVP 12 7 41.3 211.7 2.4  49.2  32.9 16.6 19 724 VAVLAVLPALAP 12 8 57.3 203.3 2.3  47.5  31.2 16.1 20 563 ALAVIVVPALAP 12 8 50.2 203.2 2.4  47.1  31.4 15.9 21 811 AVVLAVPALAVP 12 7 57.3 195.0 2.3  46.5  31.1 15.7 22 831 IIVAVAPAAIVP 12 7 43.2 203.3 2.5  46.3  31.0 15.7 23 829 AALALVAPVIVP 12 8 50.2 203.3 2.4  44.8  30.0 15.2 24 891 ILAVAAIPAALP 12 a 54.9 195.2 2.2  44.7  29.9 15.1 25 905 AVIAVAPLVVAP 12 7 41.3 195.0 2.4  44.0  29.5 14.9 26 564 VAIALIVPALAP 12 8 50.2 211.7 2.4  43.6  29.1 14.7 27 124 IAVALPALIAAP 12 6 60.3 196.2 2.2  43.6  29.0 14.7 28 827 IAAVLAAPALVP 12 8 57.3 187.5 2.2  43.0  28.8 14.6 29   2 AAAVPLLAVVVP 12 5 41.3 195.0 2.4  40.9  27.2 13.2 30 385 IVAIAVPALVAP 12 7 50.2 203.3 2.4  38.8  25.9 13.1 31 828 IALLAAPIIAVP 12 7 41.3 220.0 2.4  36.8  24.6 12.4 32 806 LVALAVPAAVLP 12 7 57.3 203.3 2.3  36.7  24.6 12.4 33 845 AAVVIAPLLAVP 12 7 41.3 203.3 2.4  35.8  24.0 12.1 34 882 AIALVVPAVAVP 12 7 57.3 195.0 2.4  35.0  23.4 11.8 35 545 VVLVLAAPAAVP 12 8 57.3 196.0 2.3  34.6  23.1 11.7 36 161 AVIALPALIAAP 12 6 57.3 195.2 2.2  34.5  23.0 11.6 37 481 AIAIAIVPVALP 12 a 50.2 211.6 2.4  34.3  23.0 11.6 38 900 ALVAVIAPVVAP 12 8 57.3 195.0 2.4  34.3  22.9 11.6 39 223 AILAVPIAVVAP 12 6 57.3 203.3 2.4  33.0  22.1 11.2 40 824 LIIVAAAPAVAP 12 8 50.2 187.5 2.3  32.8  21.9 11.1 41 562 ALIAAIVPALVP 12 8 50.2 211.7 2.4  32.7  21.2 11.0 42 222 ALLIARAAVIAP 12 6 57.3 195.2 2.2  32.6  21.7 11.0 43  61 VAALPVLLAALP 12 5 57.3 211.7 2.3  31.2  20.2 10.5 44 582 VAVALIVPALAP 12 8 50.2 203.3 2.4  30.6  20.4 10.3 45 889 ILVAAAPIAALP 12 7 57.3 195.8 2.2  30.3  20.3 10.3 46 787 AVALVPVIVAAP 12 6 50.2 195.0 2.4  29.3  19.6  9.9 47 703 IVAVALVPALAP 12 8 50.2 203.3 2.4  29.2  19.5  9.9 48 705 IVAVALLPALAP 12 8 50.2 211.7 2.4  28.6  19.1  9.7 49 885 LVAIAPAVAVLP 12 6 57.3 203.3 2.4  28.3  19.0  9.6 50   3 AALLVPAAVLAP 12 6 57.3 187.5 2.1  27.0  18.0  9.1 51 601 AAILIAVPIAAP 12 8 57.3 195.8 2.3  26.8  17.9  9.0 52 843 AVLVLVAPAAAP 12 8 41.3 219.2 2.5  26.4  17.7  8.9 53 403 AAALVIPAAILP 12 7 54.9 195.8 2.2  25.2  16.8  8.5 54 544 IVALIVAPAAVP 12 8 43.1 203.3 2.4  23.4  15.6  7.9 55 522 ALLVIAVPAVAP 12 8 57.3 203.3 2.4  22.7  15.2  7.7

TABLE 27 Sequence Proline Rigidity/ Sturctural Relative ID Position Flexibility Feature Hydropathy Ratio (Fold) Number aMTD Sequences Length (PP) (II) (AI) (GRAVY) A B C  56 805 LVLIAAAPIALP 12 8 41.3 220.0 2.4 22.3 14.9 7.6  57 464 AVVILVPLAAAP 12 7 57.3 203.3 2.4 22.3 14.9 7.5  58 405 LAAAVIPVAILP 12 7 54.2 211.7 2.4 22.2 14.8 7.5  59 747 VALLAIAPALAP 12 8 57.3 195.2 2.2 22.0 14.8 7.5  60 501 VIVALAVPALAP 12 8 50.2 203.3 2.4 21.5 14.4 7.3  61 661 AAILAPIVAALP 12 6 50.2 195.2 2.2 21.4 14.3 7.2  62 786 LVAIAPLAVLAP 12 6 41.3 211.7 2.4 21.2 14.2 7.2  63 625 ILAAAAAPLIVP 12 8 50.2 195.2 2.2 20.9 13.9 7.0  64 442 ALAALVPAVLVP 12 7 57.3 203.3 2.3 20.4 13.6 6.9  65 912 VALLAPAVVVAP 12 6 57.3 195.0 2.3 19.9 13.3 6.7  66 165 ALAVPVALAIVP 12 5 50.2 203.3 2.4 19.8 13.2 6.7  67 422 VVAILAPLLAAP 12 7 57.3 211.7 2.4 19.6 13.1 6.6  68 686 AALVAVLPVALP 12 8 57.3 203.3 2.3 19.5 13.1 6.6  69 343 IVAVALPALVAP 12 7 50.2 203.3 2.3 19.4 12.9 6.5  70 323 IVAVALPVALAP 12 7 50.2 203.3 2.3 19.1 12.8 6.4  71 461 IAAVIVPAVALP 12 7 50.2 203.3 2.4 19.0 12.7 6.4  72 21 AVALLPALLAVP 12 6 57.3 211.7 2.3 18.9 12.6 6.4  73 404 LAAAV1PAA1LP 12 7 54.9 195.2 2.2 18.9 12.6 6.4  74 261 LVLVPLLAAAAP 12 5 41.3 211.6 2.3 18.5 12.3 5.2  75 524 AVALIVVPALAP 12 8 50.2 203.3 2.4 18.3 12.2 6.2  76 225 VAALLPAAAVLP 12 6 57.3 187.5 2.1 18.3 12.2 6.2  77 264 LAAAPVVIVIAP 12 5 50.2 203.3 2.4 18.2 12.1 6.1  78   1 AAALAPVVLALP 12 6 57.3 187.5 2.1 17.7 11.8 6.0  79 382 AAALVIPAILAP 12 7 54.9 195.2 2.2 17.7 11.8 6.0  80 463 AVAILVPLLAAP 12 7 57.3 211.7 2.4 17.6 11.7 5.9  81 322 VVAIVLPALAAP 12 7 50.2 203.3 2.3 17.6 11.7 5.9  82 503 AAIIIVLPAALP 12 8 50.2 220.0 2.4 17.6 11.8 5.9  83 870 VLVAAVLPIAAP 12 8 41.3 203.3 2.4 16.6 11.1 5.6  84 241 AAAVVPVLLVAP 12 6 57.3 195.0 2.4 16.6 11.0 5.6 85 726 LAVAIIAPAVAP 12 8 57.3 187.5 2.2 16.5 11.0 5.6  86 341 IVAVALPAVLAP 12 7 50.2 203.3 2.3 16.4 10.9 5.5  87 542 ALALIIVPAVAP 12 s 50.2 211.6 2.4 16.2 10.8 5.5  88 361 AVVIVAPAVIAP 12 7 50.2 195.0 2.4 16.0 10.7 5.4  89 224 ILAAVPIALAAP 12 6 57.3 195.8 2.2 15.8 10.6 5.3  90 482 ILAVAAIPVAVP 12 s 54.9 203.3 2.4 15.8 10.6 5.3  91  64 AIVALPVAVLAP 12 6 50.2 203.3 2.4 15.8 10.6 5.3  92 484 LAVVLAAPAIVP 12 8 50.2 203.3 2.4 15.6 10.4 5.3  93 868 VLVAAILPAAIP 12 8 54.2 211.7 2.4 14.9 10.0 5.0  94 541 LLALIIAPAAAP 12 8 57.3 204.1 2.1 14.2  9.9 5.0  95 666 AAIAIIAPAIVP 12 8 50.2 195.8 2.3 14.7  9.9 5.0  96 665 LAIVLAAPVAVP 12 8 50.2 203.3 2.3 14.7  9.9 5.0  97 363 AVLAVAPALIVP 12 7 50.2 203.3 2.3 14.7  9.8 4.9  98 242 AALLVPALVAAP 12 6 57.3 187.5 2.1 14.6  9.7 4.9  99 384 VIVAIAPALLAP 12 7 50.2 211.6 2.4 14.0  9.4 4.7 100 877 VAIIAVPAVVAP 12 7 57.3 195.0 2.4 14.0  9.4 4.7 101 863 AAVVLLPIIAAP 12 7 41.3 211.7 2.4 13.8  9.3 4.7 102 525 ALAIVVAPVAVP 12 8 50.2 195.0 2.4 13.8  9.2 4.7 103 875 AIAIVVPAVAVP 12 7 50.2 195.0 2.4 13.8  9.2 4.7 104 285 AIVLLPAAVVAP 12 6 50.2 203.3 2.4 13.3  8.9 4.5 105 281 ALIVLPAAVAVP 12 6 50.2 203.3 2.4 13.3  8.9 4.5 106 867 ALLVVIAPLAAP 12 8 41.3 211.7 2.4 13.2  8.8 4.4 107 766 IVVIAVAPAVAP 12 8 50.2 195.0 2.4 12.9  8.6 4.4 108 342 VIVALAPAVLAP 12 7 50.2 203.3 2.3 12.7  8.5 4.3 109 881 AALIVVPAVAVP 12 7 50.2 195.0 2.4 12.7  8.5 4.3 110 505 AIIIVIAPAAAP 12 8 50.2 195.8 2.3 12.4  8.3 4.2

TABLE 28 Sequence Proline Rigidity/ Sturctural Relative ID Position Flexibility Feature Hydropathy Ratio (Fold) Number aMTD Sequences Length (PP) (II) (AI) (GRAVY) A B C 111 763 VAVLIAVPALAP 12 8 57.3 203.3 2.3 12.3 7.2 4.2 112 706 IVAVALLPAVAP 12 8 50.2 203.3 2.4 12.0 7.0 4.1 113 687 AILAVALPLLAP 12 8 57.3 220.0 2.3 12.0 7.0 4.1 114 643 LALVLAAPAIVP 12 8 50.2 211.6 2.4 11.8 7.9 4.0 115 282 VLAVAPALIVAP 12 6 60.2 203.3 2.4 11.8 7.9 4.0 116 543 LLAALIAPAALP 12 8 57.3 204.1 2.1 11.7 7.8 4.0 117 325 IVAVALPAVALP 12 7 50.2 203.3 2.3 11.7 7.8 4.0 118 846 IAVAVAAPLLVP 12 8 41.3 203.3 2.4 11.7 6.8 4.0 119 383 VIVALAPALLAP 12 7 50.2 211.6 2.3 11.6 7.7 3.9 120 381 VVAIVLPAVAAP 12 7 50.2 195.0 2.4 11.3 7.7 3.9 121 808 LVVLAAAPLAVP 12 8 41.3 203.3 2.3 11.5 7.6 3.9 122 865 AVLVIAVPAIAP 12 8 57.3 203.3 2.5 11.3 7.5 3.8 123 725 IAVLAVAPAVLP 12 8 67.3 203.3 2.3 11.2 7.5 3.8 124 844 VVALLAPLIAAP 12 7 41.3 211.8 2.4 11.2 7.5 3.8 125 897 AV1VPVAIIAAP 12 5 50.2 203.3 2.5 11.2 7.5 3.8 126 605 VIAAVLAPVAVP 12 8 57.3 195.0 2.4 11.0 7.4 3.7 127 744 AAVVIVAPVALP 12 8 50.2 195.0 2.4 11.0 7.3 3.7 128 221 AAILAPIVALAP 12 6 50.2 195.8 2.2 10.9 7.3 3.7 129 622 ALIVLAAPVAVP 12 8 60.2 203.3 2.4 10.6 7.1 3.6 130 401 AALAVIPAAILP 12 7 54.9 195.8 2.2 10.6 7.1 3.6 131 324 IVAVALPAALVP 12 7 50.2 203.3 2.3 10.3 6.9 3.5 132 878 IVALVAPAAVVP 12 7 50.2 195.0 2.4 10.3 6.9 3.5 133 302 LALAPALALLAP 12 5 57.3 204.2 2.1 10.2 6.8 3.4 134 685 ALLVAVLPAALP 12 8 57.3 211.7 2.3 10.2 5.9 3.4 135 848 AVAIVVLPAVAP 12 8 50.2 195.0 2.4 10.0 6.7 3.4 136 602 VIVALAAPLAP 12 8 50.2 203.3 2.4  9.9 6.8 3.4 137 788 AIAVAIAPVALP 12 8 57.3 187.5 2.3  9.8 6.6 3.3 138 145 LLAVVPAVALAP 12 6 57.3 203.3 2.3  9.5 6.3 3.2 139  11 VVALAPALAALP 12 6 57.3 187.5 2.1  9.5 6.3 3.2 140 141 AVIVLPALAVAP 12 6 50.2 203.3 2.4  9.4 6.3 3.2 141 521 LAALIVVPAVAP 12 8 50.2 203.3 2.4  9.4 6.3 3.2 142 425 AVVAIAPVLALP 12 7 57.3 203.3 2.4  9.4 6.3 3.2 143 365 AVIVVAPALLAP 12 7 50.2 203.3 2.3  9.3 6.2 3.1 144 263 ALAVIPAAAILP 12 6 54.9 195.8 2.2  9.0 6.0 3.0 145 345 ALLIVAPVAVAP 12 7 50.2 203.3 2.3  8.9 5.9 3.0 146 850 LVIALAAPVALP 12 8 57.3 211.7 2.4  8.8 5.9 3.0 147 144 VLAIVPAVALAP 12 6 50.2 203.3 2.4  8.8 5.9 3.0 148 767 IVVAAVVPALAP 12 8 50.2 195.0 2.4  8.5 5.0 2.9 149 185 AALVLPLIIAAP 12 6 41.3 220.0 2.4  8.5 5.7 2.9 150 849 AVILLAPLIAAP 12 7 57.3 220.0 2.4  8.3 4.8 2.8 151 864 ALLVIAPAIAVP 12 7 57.3 211.7 2.4  8.2 4.8 2.8 152 162 AVVALPAALIVP 12 6 50.2 203.3 2.4  8.2 5.5 2.8 153 164 LAAVLPALLAAP 12 6 57.3 195.8 2.1  8.2 5.5 2.8 154 907 VAIALAPVVVAP 12 7 57.3 195.0 2.4  8.1 5.4 2.8 155 444 LAPALVPVALVP 12 7 57.3 203.3 2.3  8.1 5.4 2.7 156 443 ALAALVPVALVP 12 7 57.3 203.3 2.3  8.0 5.3 2.7 157 901 ALVAVLPAVAVP 12 7 57.3 195.0 2.4  7.7 5.1 2.6 158 887 VLAVAPAVAVLP 12 6 57.3 195.0 2.4  7.7 5.1 2.6 159 746 VAIIVVAPALAP 12 8 50.2 203.3 2.4  7.6 4.4 2.6 160 902 ALVAPLLAVAVP 12 5 41.3 203.3 2.3  7.6 5.1 2.6 161 565 VAIVLVAPAVAP 12 8 50.2 195.0 2.4  7.5 5.0 2.5 162 245 AAALAPVLALVP 12 6 57.3 187.5 2.1  7.5 5.0 2.5 163 743 AIAIALVPVALP 12 8 57.3 211.6 2.4  7.4 4.9 2.5 164 465 AVVILVPLAAAP 12 7 57.3 203.3 2.4  7.4 4.9 2.5 165 104 AVVAAPLVLALP 12 6 41.3 203.3 2.3  7.3 4.9 2.5

TABLE 29 Sequence Proline Rigidity/ Sturctural Relative ID Position Flexibility Feature Hydropathy Ratio (Fold) Number aMTD Sequences Length (PP) (II) (AI) (GRAVY) A B C 166 707 IVALAVLPAVAP 12 8 50.2 203.3 2.4 7.3 4.9 2.5 167 872 VLAAAVLPLVVP 12 8 41.3 219.2 2.5 7.3 4.9 2.5 168 583 AVILALAPIVAP 12 8 50.2 211.6 2.4 7.3 4.8 2.4 169 87$ AAIVLLPAVVVP 12 7 50.2 219.1 2.5 7.2 4.8 2.4 170 784 VAALPAVALVVP 12 5 57.3 195.0 2.4 7.1 4.7 2.4 171 893 VIAIPAILAAAP 12 5 54.9 195.8 2.3 7.0 4.7 2.4 172  13 AAALVPVVALLP 12 6 57.3 203.3 2.3 7.0 4.7 2.4 173 809 LIVLAAPALAAP 12 7 50.2 195.2 2.2 7.0 4.7 2.4 174 445 ALAALVPALVVP 12 7 57.3 203.3 2.3 6.9 4.6 2.3 175  81 AALLPALAALLP 12 5 57.3 204.2 2.1 6.9 4.5 2.3 176 667 LAVAIVAPALVP 12 8 50.2 203.3 2.3 6.9 4.6 2.3 177 906 AV1ALAPVVVAP 12 7 57.3 195.0 2.4 6.8 4.6 2.3 178 483 ILAAAIIPAALP 12 8 54.9 204.1 2.2 6.8 4.5 2.3 179 485 AILAAIVPLAVP 12 8 50.2 211.6 2.4 6.8 4.5 2.3 180 421 AAILAAPLIAVP 12 7 57.3 195.2 2.2 6.7 4.5 2.3 181 585 ALIVAIAPALVP 12 8 50.2 211.6 2.4 6.6 4.4 2.2 182 424 AVVVAAPVLALP 12 7 57.3 195.0 2.4 6.6 4.4 2.2 183 364 LVAAVAPALIVP 12 7 50.2 203.3 2.3 6.5 4.3 2.2 184 402 ALAAVIPAAILP 12 7 54.9 195.2 2.2 6.4 4.3 2.2 185 462 IAAVLVPAVALP 12 7 57.3 203.3 2.4 6.3 4.2 2.1 186 265 VLAIAPLLAAVP 12 6 41.3 211.6 2.3 6.0 4.0 2.0 187 301 VlAAPVLAVLAP 12 6 57.3 203.3 2.4 6.0 4.0 2.0 188 183 LLAAPVVIALAP 12 6 57.3 211.6 2.4 6.0 4.0 2.0 189 243 AAVLLPVALAAP 12 6 57.3 187.5 2.1 5.9 3.9 2.0 190 664 ILIAIAIPAAAP 12 8 54.9 204.1 2.3 5.7 3.2 1.9 191 783 IVALVPAVAIAP 12 6 50.2 203.3 2.5 5.7 3.8 1.9 192 502 AIVALAVPVLAP 12 8 50.2 203.3 2.4 5.6 3.7 1.9 193 262 ALIAVPAIIVAP 12 6 50.2 211.6 2.4 5.5 3.7 1.9 194 683 LAIVLAAPAVLP 12 8 50.2 211.7 2.4 5.5 3.2 1.9 195 830 IALVAAPVALVP 12 7 57.3 203.3 2.4 5.3 3.5 1.8 196 764 AVALAVLPAVVP 12 8 57.3 195.0 2.3 5.0 3.4 1.7 197 807 AVALAVPALVLP 12 7 57.3 203.3 2.3 5.0 3.3 1.7 198 184 LAAIVPAIIAVP 12 6 50.2 211.6 2.4 4.8 3.2 1.6 199 305 IALAAPILLAAP 12 6 57.3 204.2 2.2 4.8 3.2 1.6 200 101 LVALAPVAAVLP 12 6 57.3 203.3 2.3 4.5 3.0 1.5 201 304 AIILAPIAAIAP 12 6 57.3 204.2 2.3 4.4 3.0 1.5 202 604 VALIAVAPAVVP 12 8 57.3 195.0 2.4 4.3 2.5 1.5 203 645 ALAVVALPAIVP 12 8 50.2 203.3 2.4 4.3 2.9 1.5 204 201 LALAVPALAALP 12 6 57.3 195.2 2.1 4.2 2.2 1.4 205 163 LALVLPAALAAP 12 6 57.3 195.2 2.1 4.1 2.4 1.4 206 832 AVAAIVPVIVAP 12 7 43.2 195.0 2.5 4.1 2.7 1.4 207 182 ALIAPVVALVAP 12 6 57.3 203.3 2.4 4.0 2.7 1.4 208  23 VVLVLPAAAAVP 12 6 57.3 195.0 2.4 4.0 2.2 1.3 209 105 LLALAPAALLAP 12 6 57.3 204.1 2.1 4.0 2.6 1.3 210 561 AAVAIVLPAVVP 12 8 50.2 195.0 2.4 3.9 2.6 1.3 211 765 AVALAVVPAVLP 12 8 57.3 195.0 2.3 3.8 2.2 1.3 212 684 AAIVLALPAVLP 12 8 50.2 211.7 2.4 3.5 2.1 1.2 213 143 AVLAVPAVLVAP 12 6 57.3 195.0 2.4 3.3 2.2 1.1 214 504 LIVALAVPALAP 12 8 50.2 211.7 2.4 3.3 2.2 1.1 215  22 AVVLVPVLAAAP 12 6 57.3 195.0 2.4 3.1 2.1 1.1 216   5 AAALLPVALVAP 12 6 57.3 187.5 2.1 3.1 2.1 1.0 217 283 AALLAPALIVAP 12 6 50.2 195.8 2.2 3.1 2.0 1.0 218  65 IAIVAPVVALAP 12 6 50.2 203.3 2.4 3.0 2.0 1.0 219 883 LAIVPAAIAALP 12 6 50.2 195.8 2.2 3.0 2.0 1.0 220 123 AAIIVPAALLAP 12 6 50.2 195.8 2.2 2.9 2.0 1.0

TABLE 30 Rigidity/ Sturc- Sequence Proline Flexi- tural Hydro- Relative ID Position bility Feature pathy Ratio (Fold) Number aMTD Sequences Length (PP) (II) (AI) (GRAVY) A B C 221 284 ALIAPAVALIVP 12 5 50.2 211.7 2.4 2.8 1.8 0.9 222 205 ALALVPAIAALP 12 6 57.3 195.2 2.2 2.6 1.7 0.9 223  42 VAALPVVAVVAP 12 5 57.2 186.7 2.4 2.5 1.7 0.8 224 121 AIVALPALALAP 12 6 50.2 195.8 2.2 2.5 1.7 0.8 225  25 IVAVAPALVALP 12 6 50.2 203.3 2.4 2.4 1.6 0.8 226  24 IALAAPALIVAP 12 6 50.2 195.8 2.2 2.3 1.6 0.8 227 204 LIAALPAVAALP 12 6 57.3 195.2 2.2 2.2 1.5 0.8 228  12 LLAAVPAVLLAP 12 6 57.2 211.7 2.3 2.2 1.5 0.7 229  43 LLAAPLVVAAVP 12 5 41.3 187.5 2.1 2.1 1.4 0.7 236 103 ALIAAPILALAP 12 6 57.3 204.2 2.2 2.1 1.4 0.7 231  82 AVVLAPVAAVLP 12 6 57.2 195.0 2.4 2.1 1.4 0.7 232   4 ALALLPVAALAP 12 6 57.2 195.8 2.1 2.0 1.3 0.7 233  85 LLVLPAAALAAP 12 5 57.3 195.8 2.1 1.9 1.3 0.7 234  63 AALLVPALVAVP 12 6 57.3 203.3 2.3 1.9 1.3 0.7 235  44 ALAVPVALLVAP 12 5 57.3 203.3 2.3 1.6 1.1 0.5 236  84 AAVAAPLLLALP 12 6 41.3 195.8 2.1 1.5 1.0 0.5 237  62 VALLAPVALAVP 12 6 57.3 203.3 2.3 1.4 0.9 0.5 238  83 LAVAAPLALALP 12 6 41.3 195.8 2.1 1.4 0.9 0.5 239 102 LALAPAALALLP 12 5 57.3 204.2 2.1 1.4 0.9 0.5 240 623 VAAAIALPAIVP 12 8 50.2 187.5 2.3 0.8 0.6 0.3 19.6 ± 1.6 13.1 ± 11 6.6 ± 0.5

Moreover, compared to reference CPPs (B type: MTM12 and C type: MTD85), novel 240 aMTDs averaged of 13±1.1 (maximum 109.9) and 6.6±0.5 (maximum 55.5) fold higher cell-permeability, respectively (Tables 26-31).

TABLE 31 Negative Control rP38 MTM12 MTD85 aMTD 19.6 ± 1.6* 13.1 ± 1.1* 6.6 ± 0.5* The Average of 240 aMTDs (Best: 164.2) (Best: 109.9) (Best: 55.5) *Relative Fold (aMTD in Geo Mean in its comparison to rP38, MTM12 or MTD85)

In addition, cell-permeability of 31 rPeptides has been compared with that of 240 aMTDs (0.3±0.04; Tables 32 and 33).

TABLE 32 Relative Proline Rigidity/ Sturctural Ratio Position Flexibility Feature Hydropathy to aMTD Number ID Sequence Length (PP) (II) (AI) (GRAVY) AVE  1 692 PAPLPPVNALAV 12 1,3,5,6 105.5  186.7  1.8 0.74  2  26 AAIALAAPLAIV 12 8  18.1  204.2  2.5 0.65  3 113 PVAVALLIAVPP 12 1,11,12  57.3  195.0  2.1 0.61  4 466 IIAAAAPLAIIP 12 7,12  22.8  204.2  2.3 0.52  5 167 VAIAIPAALAIP 12 6,12  20.4  195.8  2.3 0.50  6  97 ALLAAPPALLAL 12 6,7  57.3  204.2  2.1 0.41  7 390 VPLLVPVVPVVP 12 2,6,9,12 105.4  210.0  2.2 0.41  8 426 AAALAIPLAIIP 12 7,12   4.37 204.2  2.2 0.40  9 214 ALIVAPALMALP 12 6,12  60.5  187.5  2.2 0.33 10  68 VAPVLPAAPLVP 12 3,6,9,12 105.5  162.5  1.6 0.32 11  39 CYNTSPCTGCCY 12 6  52.5    0.0  0.0 0.29 12 934 LILAPAAVVAAA 12 5  57.3  195.8  2.5 0.28 13 938 VPVLLPVVVPVP 12 2,6,10,12 121.5  210.0  2.2 0.28 14 329 LPVLVPVVPVVP 12 2,6,9,12 121.5  210.0  2.2 0.23 15 606 AAAIAAIPIIIP 12 8,12   4.4  204.2  2.4 0.20 16  49 VVPAAPAVPVVP 12 3,6,9,12 121.5  145.8  1.7 0.18 17 139 TGSTNSPTCTST 12 7  53.4    0.0 -0.7 0.17 18 772 LPVAPVIPIIVP 12 2,5,8,12  79.9  210.8  2.1 0.16 19 921 IWWFVVLPLVVP 12 8,12  41.3  194.2  2.2 0.14 20  66 AGVLGGPIMGVP 12 7,12  35.5  121.7  1.3 0.13 21 693 AAPVLPVAVPIV 12 3,6,10  82.3  186.7  2.1 0.13 22  18 NYCCTPTTNGQS 12 6  47.9    0.0 -0.9 0.10 23  16 NNSCTTYTNGSQ 12 None  47.4    0.0 -1.4 0.08 24 227 LAAIVPIAAAVP 12 6,12  34.2  187.5  2.2 0.08 25  17 GGCSAPQTTCSN 12 6  51.6    8.3 -0.5 0.08 26  67 LDAEVPLADDVP 12 6,12  34.2  130.0  0.3 0.08 27 635 GSTGGSQQNNQY 12 None  31.9    0.0 -1.9 0.07 28  29 VLPPLPVLPVLP 12 3,4,6,9,12 121.5  202.5  1.7 0.07 29  57 QNNCNTSSQGGG 12 None  52.4    0.0 -1.6 0.06 30 700 GTSNTCQSNQNS 12 None  19.1    0.0 -1.6 0.05 31  38 YYNQSTCGGQCY 12 ND  53.8    0.0 -1.0 0.05 AVE 0.3 ± 0.04

TABLE 33 Relative Ratio to aMTD AVE* rPeptide 0.3 ± 0.04 The Average of 31 aMTDs *Out of 240 aMTDs, average relative fold of aMTD had been 19.6 fold compared to type A (rP38).

In summary, relative cell-permeability of aMTDs has shown maximum of 164.0, 109.9 and 55.5 fold higher to rP38, MTM12 and MTD85, respectively. In average of total 240 aMTD sequences, 19.6±1.6, 13.1±1.1 and 6.6±0.5 fold higher cell-permeability are shown to the rP38, MTM12 and MTD85, respectively (Tables 26-31). Relative cell-permeability of negative control (rP38) to the 240 aMTDs is only 0.3±0.04 fold.

4-5. Intracellular Delivery and Localization of aMTD-Fused Recombinant Proteins

Recombinant proteins fused to the aMTDs were tested to determine their intracellular delivery and localization by laser scanning confocal microscopy with a negative control (rP38) and previous published CPPs (MTM12 and MTD85) as the positive control references. NIH3T3 cells were exposed to 10 μM of FITC-labeled protein for 1 hour at 37, and nuclei were counterstained with DAPI. Then, cells were examined by confocal laser scanning microscopy (FIG. 7). Recombinant proteins fused to aMTDs clearly display intracellular delivery and cytoplasmic localization (FIG. 7) that are typically higher than the reference CPPs (MTM12 and MTD85). The rP38-fused recombinant protein did not show internalized fluorescence signal (FIG. 7a ). In addition, as seen in FIG. 8, rPeptides (his-tagged CRA recombinant proteins fused to each rPeptide) display lower- or non-cell-permeability.

4-6. Summary of Quantitative and Visual Cell-Permeability of Newly Developed aMTDs

Histidine-tagged aMTD-fused cargo recombinant proteins have been greatly enhanced in their solubility and yield. Thus, FITC-conjugated recombinant proteins have also been tested to quantitate and visualize intracellular localization of the proteins and demonstrated higher cell-permeability compared to the reference CPPs.

In the previous studies using the hydrophobic signal-sequence-derived CPPs-MTS/MTM or MTDs, 17 published sequences have been identified and analyzed in various characteristics such as length, molecular weight, pI value, bending potential, rigidity, flexibility, structural feature, hydropathy, amino acid residue and composition, and secondary structure of the peptides. Based on these analytical data of the sequences, novel artificial and non-natural peptide sequences designated as advanced MTDs (aMTDs) have been invented and determined their functional activity in intracellular delivery potential with aMTD-fused recombinant proteins.

aMTD-fused recombinant proteins have promoted the ability of protein transduction into the cells compared to the recombinant proteins containing rPeptides and/or reference hydrophobic CPPs (MTM12 and MTD85). According to the results, it has been demonstrated that critical factors of cell-penetrating peptide sequences play a major role to determine peptide-mediated intracellular delivery by penetrating plasma membrane. In addition, cell-permeability can considerably be improved by following the rational that all satisfy the critical factors.

5. Structure/Sequence Activity Relationship (SAR) of aMTDs on Delivery Potential

After determining the cell-permeability of novel aMTDs, structure/sequence activity relationship (SAR) has been analyzed for each critical factor in selected some of and all of novel aMTDs (FIGS. 13 to 16 and Table 34).

TABLE 34 Rank of Rigidity/ Sturctural Relative Ratio Delivery Flexibility Feature Hydropathy (Fold) Amino Acid Composition Potential (II) (Al) (GRAVY) A B C A V I L  1~10 55.9 199.2 2.3 112.7 75.5 38.1 4.0 3.5 0.4 2.1 11~20 51.2 205.8 2.4 56.2 37.6 19.0 4.0 2.7 1.7 1.6 21~30 49.1 199.2 2.3 43.6 28.9 14.6 4.3 2.7 1.4 1.6 31~40 52.7 201.0 2.4 34.8 23.3 11.8 4.2 2.7 1.5 1.6 41~50 53.8 201.9 2.3 30.0 20.0 10.1 4.3 2.3 1.1 2.3 51~60 51.5 205.2 2.4 23.5 15.7 7.9 4.4 2.1 1.5 2.0 222~231 52.2 197.2 2.3 2.2 1.5 0.8 4.5 2.1 1.0 2.4 232~241 54.1 199.7 2.2 1.7 1.2 0.6 4.6 1.7 0.2 3.5

5-1. Proline Position:

In regards to the bending potential (proline position: PP), aMTDs with its proline at 7′ or 8′ amino acid in their sequences have much higher cell-permeability compared to the sequences in which their proline position is at 5′ or 6′ (FIGS. 14a and 15a ).

5-2. Hydropathy:

In addition, when the aMTDs have GRAVY (Grand Average of Hydropathy) ranging in 2.1-2.2, these sequences display relatively lower cell-permeability, while the aMTDs with 2.3-2.6 GRAVY are shown significantly higher one (FIGS. 14b and 15b ).

5-3. rPeptide SAR:

To the SAR of aMTDs, rPeptides have shown similar SAR correlations in the cell-permeability, pertaining to their proline position (PP) and hydropathy (GRAVY). These results confirms that rPeptides with high GRAVY (2.4˜2.6) have better cell-permeability (FIG. 16).

5-4. Analysis of Amino Acid Composition:

In addition to proline position and hydropathy, the difference of amino acid composition is also analyzed. Since aMTDs are designed based on critical factors, each aMTD-fused recombinant protein has equally two proline sequences in the composition. Other hydrophobic and aliphatic amino acids—alanine, isoleucine, leucine and valine—are combined to form the rest of aMTD peptide sequences.

Alanine: In the composition of amino acids, the result does not show a significant difference by the number of alanine in terms of the aMTD's delivery potential because all of the aMTDs have three to five alanines. In the sequences, however, four alanine compositions show the most effective delivery potential (geometric mean) (FIG. 13a ).

Leucine and Isoleucine: Also, the compositions of isoleucine and leucine in the aMTD sequences show inverse relationship between the number of amino acid (I and L) and delivery potential of aMTDs. Lower number of isoleucine and leucine in the sequences tends to have higher delivery potential (geometric mean) (FIGS. 13a and 13b ).

Valine: Conversely, the composition of valine of aMTD sequences shows positive correlation with their cell-permeability. When the number of valine in the sequence is low, the delivery potential of aMTD is also relatively low (FIG. 13b ).

Ten aMTDs having the highest cell-permeability are selected (average geometric mean: 2584±126). Their average number of valine in the sequences is 3.5; 10 aMTDs having relatively low cell-permeability (average geometric mean: 80±4) had average of 1.9 valine amino acids. The average number of valine in the sequences is lowered as their cell-permeability is also lowered as shown in FIG. 13b . Compared to higher cell-permeable aMTDs group, lower sequences had average of 1.9 in their valine composition. Therefore, to obtain high cell-permeable sequence, an average of 2-4 valines should be composed in the sequence.

5-5. Conclusion of SAR Analysis:

As seen in FIG. 15, all 240 aMTDs have been examined for these association of the cell-permeability and the critical factors: bending potential (PP), rigidity/flexibility (II), structure feature (AI), and hydropathy (GRAVY), amino acid length and composition. Through this analysis, cell-permeability of aMTDs tends to be lower when their central proline position is at 5′ or 6′ and GRAVY is 2.1 or lower (FIG. 15). Moreover, after investigating 10 higher and 10 lower cell-permeable aMTDs, these trends are clearly shown to confirm the association of cell-permeability with the central proline position and hydropathy.

6. Experimental Confirmation of Index Range/Feature of Critical Factors

The range and feature of five out of six critical factors have been empirically and experimentally determined that are also included in the index range and feature of the critical factors initially proposed before conducting the experiments and SAR analysis. In terms of index range and feature of critical factors of newly developed 240 aMTDs, the bending potential (proline position: PP), rigidity/flexibility (Instability Index: II), structural feature (Aliphatic Index: AI), hydropathy (GRAVY), amino acid length and composition are all within the characteristics of the critical factors derived from analysis of reference hydrophobic CPPs.

Therefore, our hypothesis to design and develop new hydrophobic CPP sequences as advanced MTDs is empirically and experimentally proved and demonstrated that critical factor-based new aMTD rational design is correct.

TABLE 35 Summarized Critical Factors of aMTD Newly Designed Analysis of CPPs Experimental Results Critical Factor Range Range Bending Potential Proline presences in the Proline presences in the (Proline Position: middle (5′, 6′, 7′ or 8′) middle (5′, 6′, 7′ or 8′) PP) and at the end and at the end of peptides of peptides Rigidity/Flexibility 40-60 41.3-57.3 (Instability Index: II) Structural Feature 180-220 187.5-220.0 (Aliphatic Index: AI) Hydropathy 2.1-2.6 2.2-2.6 (Grand Average of Hydropathy GRAVY) Length  9-13 12 (Number of Amino Acid) Amino acid A, V, I, L, P A, V, I, L, P Composition 7. Discovery and Development of Protein-Based New Biotherapeutics with MITT Enabled by aMTDs for Protein Therapy

Total of 240 aMTD sequences have been designed and developed based on the critical factors. Quantitative and visual cell-permeability of 240 aMTDs (hydrophobic, flexible, bending, aliphatic and 12 a/a-length peptides) are all practically determined.

To measure the cell-permeability of aMTDs, rPeptides have also been designed and tested. As seen in FIGS. 13 to 15, there are vivid association of cell-permeability and the critical factors of the peptides. Out of these critical factors, we are able to configure that the most effective cell-permeable aMTDs have the amino acid length of 12; composition of A, V, L, I and P; multiple proline located at either 7′ or 8′ and at the end (12′); instability index ranged of 41.3-57.3; aliphatic index ranged of 187.5-220.0; and hydropathy (GRAVY) ranged of 2.2-2.6.

These examined critical factors are within the range that we have set for our critical factors; therefore, we are able to confirm that the aMTDs that satisfy these critical factors have relatively high cell-permeability and much higher intracellular delivery potential compared to reference hydrophobic CPPs reported during the past two decades.

It has been widely evident that many human diseases are caused by proteins with deficiency or over-expression that causes mutations such as gain-of-function or loss-of-function. If biologically active proteins could be delivered for replacing abnormal proteins within a short time frame, possibly within an hour or two, in a quantitative manner, the dosage may be regulated depending on when and how proteins may be needed. By significantly improving the solubility and yield of novel aMTD in this invention (Table 31), one could expect its practical potential as an agent to effectively deliver therapeutic macromolecules such as proteins, peptides, nucleic acids, and other chemical compounds into live cells as well as live mammals including human. Therefore, newly developed MITT utilizing the pool (240) of novel aMTDs can be used as a platform technology for discovery and development of protein-based biotherapeutics to apprehend intracellular protein therapy after determining the optimal cargo-aMTD relationship.

The following examples are presented to aid practitioners of the invention, to provide experimental support for the invention, and to provide model protocols. In no way are these examples to be understood to limit the invention.

Example 1. Development of Novel Advanced Macromolecule Transduction Domain (aMTD)

H-regions of signal sequences (HRSP)-derived CPPs (MTS/MTM and MTD) do not have a common sequence, a sequence motif, and/or a common structural homologous feature. In this invention, the aim is to develop improved hydrophobic CPPs formatted in the common sequence and structural motif that satisfy newly determined ‘critical factors’ to have a ‘common function,’ to facilitate protein translocation across the plasma membrane with similar mechanism to the analyzed CPPs.

The structural motif as follows:

In Table 9, universal common sequence/structural motif is provided as follows. The amino acid length of the peptides in this invention ranges from 9 to 13 amino acids, mostly 12 amino acids, and their bending potentials are dependent with the presence and location of proline in the middle of sequence (at 5′, 6′, 7′ or 8′ amino acid) and at the end of peptide (at 12′) for recombinant protein bending. Instability index (II) for rigidity/flexibility of aMTDs is II<40, grand average of hydropathy (GRAVY) for hydropathy is around 2.2, and aliphatic index (AI) for structural features is around 200 (Table 9). Based on these standardized critical factors, new hydrophobic peptide sequences, namely advanced macromolecule transduction domain peptides (aMTDs), in this invention have been developed and summarized in Tables 10 to 15.

Example 2. Construction of Expression Vectors for Recombinant Proteins Fused to aMTDs

Our newly developed technology has enabled us to expand the method for making cell-permeable recombinant proteins. The expression vectors were designed for histidine-tagged CRA proteins fused with aMTDs or rPeptides. To construct expression vectors for recombinant proteins, polymerase chain reaction (PCR) had been devised to amplify each designed aMTD or rPeptide fused to CRA.

The PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, 1× reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctor protein, Korea) was digested on the restriction enzyme site between Nde I (5′) and Sal I (3′) involving 35 cycles of denaturation (95° C.), annealing (62° C.), and extension (72° C.) for 30 seconds each. For the last extension cycle, the PCR reactions remained for 5 minutes at 72° C. Then, they were cloned into the site of pET-28a(+) vectors (Novagen, Darmstadt, Germany). DNA ligation was performed using T4 DNA ligase at 4° C. overnight. These plasmids were mixed with competent cells of E. coli DH5-alpha strain on the ice for 10 minutes. This mixture was placed on the ice for 2 minutes after it was heat shocked in the water bath at 42° C. for 90 seconds. Then, the mixture added with LB broth media was recovered in 37° C. shaking incubator for 1 hour. Transformant was plated on LB broth agar plate with kanamycin (50 μg/mL) (Biopure, Johnson City, Tenn., USA) before incubating at 37° C. overnight. From a single colony, plasmid DNA was extracted, and after the digestion of Nde I and Sal I restriction enzymes, digested DNA was confirmed at 645 bp by using 1.2% agarose gels electrophoresis (FIG. 2). PCR primers for the CRA recombinant proteins fused to aMTD and random peptides (rPeptide) are summarized in Tables 23 to 30. Amino acid sequences of aMTD and rPeptide primers are shown in Tables 31 to 38.

Example 3. Inducible Expression, Purification and Preparation of Recombinant Proteins Fused to aMTDs and rPeptides

To express recombinant proteins, pET-28a(+) vectors for the expression of CRA proteins fused to a negative control [rPeptide 38 (rP38)], reference hydrophobic CPPs (MTM₁₂ and MTD₈₅) and aMTDs were transformed in E. coli BL21 (DE3) strains. Cells were grown at 37° C. in LB medium containing kanamycin (50 μg/ml) with a vigorous shaking and induced at OD₆₀₀=0.6 by adding 0.7 mM IPTG (Biopure) for 2 hours at 37° C. Induced recombinant proteins were loaded on 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue (InstantBlue, Expedeon, Novexin, UK) (FIG. 3).

The E. coli cultures were harvested by centrifugation at 5,000×rpm for 10 minutes, and the supernatant was discarded. The pellet was re-suspended in the lysis buffer (50 mM NaH₂PO₄, 10 mM Imidazol, 300 mM NaCl, pH 8.0). The cell lysates were sonicated on ice using a sonicator (Sonics and Materials, Inc., Newtown, Conn., USA) equipped with a probe. After centrifuging the cell lysates at 5,000×rpm for 10 minutes to pellet the cellular debris, the supernatant was incubated with lysis buffer-equilibrated Ni-NTA resin (Qiagen, Hilden, Germany) gently by open-column system (Bio-rad, Hercules, Calif., USA). After washing protein-bound resin with 200 ml wash buffer (50 mM NaH₂PO₄, 20 mM Imidazol, 300 mM NaCl, pH 8.0), the bounded proteins were eluted with elution buffer (50 mM NaH₂PO₄, 250 mM Imidazol, 300 mM NaCl, pH 8.0).

Recombinant proteins purified under natural condition were analyzed on 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue (FIG. 4). All of the recombinant proteins were dialyzed for 8 hours and overnight against physiological buffer, a 1:1 mixture of cell culture medium (Dulbecco's Modified Eagle's Medium: DMEM, Hyclone, Logan, Utah, USA) and Dulbecco's phosphate buffered saline (DPBS, Gibco, Grand Island, N.Y., USA). From 316 aMTDs and 141 rPeptides cloned, 240 aMTD- and 31 rPeptide-fused recombinant proteins were induced, purified, prepared and analyzed for their cell-permeability.

Example 4. Determination of Quantitative Cell-Permeability of Recombinant Proteins

For quantitative cell-permeability, the aMTD- or rPeptide-fused recombinant proteins were conjugated to fluorescein isothiocyanate (FITC) according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, Mo., USA). RAW 264.7 cells were treated with 10 μM FITC-labeled recombinant proteins for 1 hour at 37° C., washed three times with cold PBS, treated with 0.25% tripsin/EDTA (Sigma-Aldrich, St. Louis, Mo.) for 20 minutes at 37° C. to remove cell-surface bound proteins. Cell-permeability of these recombinant proteins were analyzed by flow cytometry (Guava, Millipore, Darmstadt, Germany) using the FlowJo cytometric analysis software (FIGS. 5 to 6). The relative cell-permeability of aMTDs were measured and compared with the negative control (rP38) and reference hydrophobic CPPs (MTM12 and MTD85) (Table 31).

Example 5. Determination of Cell-Permeability and Intracellular Localization of Recombinant Proteins

For a visual reference of cell-permeability, NIH3T3 cells were cultured for 24 hours on coverslip in 24-wells chamber slides, treated with 10 μM FITC-conjugated recombinant proteins for 1 hour at 37° C., and washed three times with cold PBS. Treated cells were fixed in 4% paraformaldehyde (PFA, Junsei, Tokyo, Japan) for 10 minutes at room temperature, washed three times with PBS, and mounted with VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, Calif., USA), and counter stained with DAPI (4′,6-diamidino-2-phenylindole). The intracellular localization of the fluorescent signal was determined by confocal laser scanning microscopy (LSM700, Zeiss, Germany; FIGS. 7 and 8).

Example 6-1. Cloning of aMTD/SD-Fused SOCS3 Recombinant Protein

Full-length cDNA for human SOCS3 (SEQ ID NO: 815) was purchased from Origene (USA). Histidine-tagged human SOCS3 proteins were constructed by amplifying the SOCS3 cDNA (225 amino acids) using primers for aMTD fused to SOCS3 cargo. The PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, 1× reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctor protein, Korea)) were digested on the restriction enzyme site between Nde I (5′) and Sal I (3′) involving 35 cycles of denaturing (95° C.), annealing (62° C.), and extending (72° C.) for 45 sec each. For the last extension cycle, the PCR reactions remained for 10 min at 72° C. The PCR products were subcloned into 6×His expression vector, pET-28a(+) (Novagen, Darmstadt, Germany). Coding sequence for SDA or SDB fused to C terminus of his-tagged aMTD-SOCS3 was cloned at BamHI (5′) and SalI (3′) in pET-28a(+) from PCR-amplified DNA segments and confirmed by DNA sequence analysis of the resulting plasmids.

TABLE 36 Recombinant Cargo SD Protein 5′ Primers 3′ Primers SOCS3 — HS3 5′-GGAATTC 5′-CCCGGAT CATATGGTCA CCTTAAAGCG CCCACAGCAA GGGCATCGTA GTTTCCCGCC CTGGTCCAGG GCC-3′ AA-3′ — HM₁₆₅S3 5′-GGAATTC 5′-CCCGGAT CATATGGCGC CCTTAAAGCG TGGCGGTGCC GGGCATCGTA GGTGGCGCTG CTGGTCCAGG GCGATTGTGC AA-3′ CGGTCACCCA CAGCAAGTTT C-3′ A HM₁₆₅S3A 5′-GGAATTC 5′-CGCGTCG CATATGGCGC ACTTACCTCG TGGCGGTGCC GCTGCACCGG GGTGGCGCTG CACGGCGATA GCGATTGTGC C-3′ CGGTCACCCA CAGCAAGTTT C-3′ B HM₁₆₅S3B 5′-GGAATTC 5′-CGCGTCG CATATGGCGC ACTTAAAGGG TGGCGGTGCC TTTCCGAAGG GGTGGCGCTG CTTGGCTATC GCGATTGTGC TT-3′ CGGTCACCCA CAGCAAGTTT C-3′ C HM₁₆₅S3C 5′-GGAATTC 5′-GCGTCGA CATATGGCGC CTTAGGCCAG TGGCGGTGCC GTTAGCGTCG GGTGGCGCTG AG-3′ GCGATTGTGC CGGTCACCCA CAGCAAGTTT C-3′ D HM₁₆₅S3D 5′-GGTTCCA 5′-GCGTCGA TATGGCGCTG CTTATTTTTT GCGGTGCCGG CTCGGACAGA TGGCGCTGGC TA-3′ AGTTGTGCCG GTCACCCACA GCAAGTTT C-3′ E HM₁₆₅S3E 5′-GGAATTC 5′-ACGCGTC CATATGGCGC GACTTAACCT TGGCGGTGCC CCAATCTGTT GGTGGCGCTG CGCGGTGAGC GCGATTGTGC CTC-3′ CGGTCACCCA CAGCAAGTTT C-3′

Example 6-2. Preparation of aMTD/SD-Fused SOCS3 Recombinant Protein

To determine a stable structure of the cell-permeable aMTD/SD-fused SOCS3 recombinant protein, a pET-28a(+) vector and an E. coli BL21-CodonPlus (DE3)-RIL were subjected to the following experiment.

Each of the recombinant expression vectors, HS3, HMS3, HMS3A, HMS3B, HMS3C, HMS3D, and HMS3E prepared in example 6-1 was transformed into E. coli BL21 CodonPlus(DE3)-RIL by a heat shock method, and then 600 ul of each was incubated in an LB medium (Biopure, Johnson City, Tenn., USA) containing 50 μg/ml of kanamycin at 37° C. for 1 hour. Thereafter, the recombinant protein gene-introduced E. coli was inoculated in 7 ml of LB medium, and then incubated at 37° C. overnight. The E. coli was inoculated in 700 ml of LB medium and incubated at 37° C. until OD₆₀₀ reached 0.6. To this culture medium, 0.6 mM of isopropyl-β-D-thiogalactoside (IPTG, Gen Depot, USA) was added as a protein expression inducer, followed by further incubation at 37° C. for 3 hours. This culture medium was centrifuged at 4° C. and 8,000 rpm for 10 minutes and a supernatant was discarded to recover a cell pellet. The cell pellet thus recovered was suspended in a lysis buffer (100 mM NaH₂PO₄, 10 mM Tris-HCl, 8 M Urea, pH 8.0), and cells were disrupted by sonication (on/off time: 30 sec/30 sec, on time 2 hours, amplify 40%), and centrifuged at 15,000 rpm for 30 min to obtain a soluble fraction and an insoluble fraction.

This insoluble fraction was suspended in a denature lysis buffer (8 M Urea, 10 mM Tris, 100 mM Sodium phosphate) and purified by Ni²⁺ affinity chromatography as directed by the supplier (Qiagen, Hilden, Germany) and refolded by dialyzing with a refolding buffer (0.55 M guanidine HCl, 0.44 M L-arginine, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 100 mM NDSB, 2 mM reduced glutathione, and 0.2 mM oxidized glutathione). After purification, the proteins were put in a SnakeSkin Dialysis Tubing bag (pore size: 10000 mw, Thermo scientific, USA) and then they were dialyzed by physiological buffer (DMEM). The strain lysate where protein expression was not induced, the strain lysate where protein expression was induced by addition of IPTG, and purified proteins were loaded on SDS-PAGE to analyze protein expression characteristics and expression levels (FIGS. 19 and 20).

As shown in FIG. 19, it was confirmed that SOCS3 recombinant proteins showed high expression levels in the BL21CodonPlus(DE3)-RIL strain. SOCS3 recombinant proteins containing aMTD₁₆₅ and solubilization domain (HM₁₆₅S3A and HM₁₆₅S3B) had little tendency to precipitate whereas recombinant SOCS3 proteins lacking a solubilization domain (HM₁₆₅S3) or lacking an aMTD and a SD (HS3) were largely insoluble. Solubility of aMTD/SD-fused SOCS3 proteins was scored on a 5 point scale compared with that of SOCS3 proteins lacking the solubilization domain.

Example 6-3. Determination of Solubility/Yield of aMTD/SD-Fused SOCS3 Recombinant Proteins According to SD Type

To determine aMTD/SD-fused SOCS3 recombinant proteins having optimal cell-permeability, solubilization domains were replaced in the same manner as in Example 6-2 to prepare 5 kinds of aMTD/SD-fused SOCS3 recombinant proteins, and their solubility/yield were measured (FIG. 19).

As shown in FIG. 19, it was confirmed that the aMTD/SD-fused SOCS3 recombinant protein prepared by fusing with SDB among the different SDs showed the highest solubility/yield. Therefore, the SDB-fused iCP-SOCS3 recombinant protein was used in the subsequent experiment.

Example 6-4. Comparison Between aMTD/SD-Fused SOCS3 Recombinant Protein and Basic CPP/SD-Fused SOCS3 Recombinant Protein

To compare the solubility/yield, cell/tissue-permeability, mechanism of cytopermeability of aMTD/SD-fused SOCS3 recombinant proteins to those of conventional basic CPP/SD-fused SOCS3 recombinant proteins, cloning, preparation, and measurement of solubility/yield of the basic CPP/SD-fused SOCS3 recombinant proteins were performed in the same manner as in Examples 6-1 to 6-3 except for a known basic CPP (TAT or PolyR) being used instead of aMTD. Sequences of amino acids and nucleotides of basic CPP, and the primers used in this example are shown in FIG. 79.

The solubility/yield of aMTD165/SD-fused SOCS3 recombinant proteins was much higher than that of TAT/SD-fused SOCS3 or PolyR/SD-fused SOCS3 recombinant proteins (FIG. 80).

Example 7-1. Cell-Permeability

To examine cell-permeability of SOCS3 recombinant protein, SOCS3 recombinant proteins were conjugated to 5/6-fluorescein isothiocyanate (FITC). RAW 264.7 (KCLB, Seoul, South Korea) (FIG. 20) or NIH3T3 cells (KCLB, Seoul, South Korea) (FIG. 21) were treated with 10 μM FITC-labeled SOCS3 recombinant proteins and cultivated for 1 hr at 37° C.

In this regard, RAW 264.7 cells were cultured in a DMEM medium containing 10% fetal bovine serum (FBS, Hyclone, USA) and 500 mg/ml of 1% penicillin/streptomycin (Hyclone, USA).

After cultivation, the cells were washed three times with ice-cold PBS (Phosphate-buffered saline, Hyclone, USA) and treated with proteinase K (10 μg/mL, SIGMA, USA) to remove surface-bound proteins, and internalized proteins were measured by flow cytometry (FlowJo cytometric analysis software, Guava, Millipore, Darmstadt, Germany). Untreated cells (gray) and equimolar concentration of unconjugated FITC (FITC only, green)-treated cells were served as control (FIG. 20). Each of NIH3T3 cells was incubated for 1 hour at 37° C. with 10 μM FITC-labeled SOCS3 protein. For nuclear staining, a mixture of VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, Calif.) and DAPI (4′,6-diamidino-2-phenylindole) was added to NIH3T3 cells, and visualized using a confocal laser microscope (LSM700, Zeiss, Germany) (FIG. 21).

As shown in FIGS. 20 and 21, SOCS3 recombinant proteins containing aMTD₁₆₅ (HM₁₆₅S3, HM₁₆₅S3A and HM₁₆₅S3B) efficiently entered the cells (FIGS. 20 and 21) and were localized to various extents in cytoplasm (FIG. 21). In contrast, SOCS3 protein containing non-aMTD (HS3) did not appear to enter cells. While all SOCS3 proteins containing aMTD₁₆₅ transduced into the cells, HM₁₆₅S3B displayed more uniform cellular distribution, and protein uptake of HM₁₆₅S3B was also very efficient.

Example 7-1-2. Comparison Between aMTD/SD-Fused SOCS3 Recombinant Protein and Basic CPP/SD-Fused SOCS3 Recombinant Protein

The cell-permeability of basic CPP/SD-fused SOCS3 recombinant proteins was assessed by the same method as used in Example 7-1 except for a known basic CPP (TAT or PolyR) being used instead of aMTD. The results of the assessment were shown in FIG. 81.

According to the results, all recombinant proteins exhibited cell-permeability. Among the proteins, aMTD/SD-fused SOCS3 recombinant protein (HM₁₆₅S3B) showed the highest cell-permeability.

Example 7-2. Tissue-Permeability

To further investigate in vivo delivery of SOCS3 recombinant proteins, ICR mice (Doo-Yeol Biotech Co. Ltd., Seoul, Korea) were intraperitoneal (IP) injected with 600 μg/head of 10 μM FITC (Fluorescein isothiocyanate, SIGMA, USA)-labeled SOCS3 proteins and sacrificed after 2 hrs. From the mice, the liver, kidney, spleen, lung, heart, and brain were removed and washed with PBS, and then placed on a dry ice, and embedded with an O.C.T. compound (Sakura). After cryosectioning at 20 μm, tissue distributions of fluorescence-labeled-SOCS3 proteins in different organs was analyzed by fluorescence microscopy (Carl Zeiss, Gottingen, Germany)(FIG. 22).

As shown in FIG. 22, SOCS3 recombinant proteins fused to aMTD₁₆₅ (HM₁₆₅S3, HM₁₆₅S3A and HM₁₆₅S3B) were distributed to a variety of tissues (liver, kidney, spleen, lung, heart and, to a lesser extent, brain). Liver showed highest levels of fluorescent cell-permeable SOCS3 since intraperitoneal administration favors the delivery of proteins to this organ via the portal circulation. SOCS3 containing aMTD₁₆₅ was detectable to a lesser degree in lung, spleen and heart. aMTD/SDB-fused SOCS3 recombinant protein (HM₁₆₅S3B) showed the highest systemic delivery of SOCS3 protein to the tissues compared to the SOCS3 containing only aMTD (HM₁₆₅S3) or aMTD/SDA (HM₁₆₅S3A) proteins. These data suggest that SOCS3 protein containing both of aMTD₁₆₅ and SDB leads to higher cell-/tissue-permeability due to the increase in solubility and stability of the protein, and it displayed a dramatic synergic effect on cell-/tissue-permeability.

Example 7-2-2. Comparison Between aMTD/SD-Fused SOCS3 Recombinant Protein and Basic CPP/SD-Fused SOCS3 Recombinant Protein

The tissue-permeability of basic CPP/SD-fused SOCS3 recombinant proteins was assessed by the same method as used in Example 7-2 except for a known basic CPP (TAT or PolyR) being used instead of aMTD. The results of the assessment were shown in FIG. 82.

According to the results, only aMTD/SD-fused SOCS3 recombinant protein (HM₁₆₅S3B) exhibited superior cell-permeability.

Example 8-1 Biological Activity Test of iCP-SOCS3—Inhibition Activity of IFN-γ-Induced STAT Phosphorylation

It was examined whether the iCP-SOCS3 recombinant proteins prepared by fusion with combinations of aMTD and SD inhibits activation of the JAK/STAT-signaling pathway.

PANC-1 Cells (KCLB, Seoul, South Korea) were treated with 10 ng/ml IFN-γ (R&D systems, Abingdon, UK) for 15 min and treated with either DMEM (vehicle) or 10 μM aMTD/SD-fused SOCS3 recombinant proteins for 2 hrs. The cells were lysed in RIPA lysis buffer (Biosesang, Seongnam, Korea) containing proteinase inhibitor cocktail (Roche, Indianapolis, Ind., USA), incubated for 15 min at 4° C., and centrifuged at 13,000 rpm for 10 min at 4° C. Equal amounts of lysates were separated on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. The membranes were blocked using 5% skim milk in TBST and for western blot analysis incubated with the following antibodies: anti-phospho-STAT1 (Cell Signaling Technology, USA) and anti-phospho-STAT3 (Cell Signaling Technology, USA), then HRP conjugated anti-rabbit secondary antibody (Santacruz).

The well-known cytokine signaling inhibitory actions of SOCS3 are inflammation inhibition through i) inhibition of IFN-γ-mediated JAK/STAT and ii) inhibition of LPS-mediated cytokine secretion. The ultimate test of cell-penetrating efficiency is a determination of intracellular activity of SOCS3 proteins transported by aMTD. Since endogenous SOCS3 are known to block phosphorylation of STAT1 and STAT3 by IFN-γ-mediated Janus kinases (JAK) 1 and 2 activation, we demonstrated whether cell-permeable SOCS3 inhibits the phosphorylation of STATs. As shown in FIG. 23, All SOCS3 recombinant proteins containing aMTD (HM₁₆₅S3, HM₁₆₅S3A and HM₁₆₅S3B), suppressed IFN-γ-induced phosphorylation of STAT1 and STAT3. In contrast, STAT phosphorylation was readily detected in cells exposed to HS3, which lacks the aMTD motif required for membrane penetration, indicating that HS3, which lacks an MTD sequence did not enter the cells, has no biological activity.

Example 8-2. Biological Activity Test of iCP-SOCS3

Peritoneal macrophages were obtained from C3H/HeJ mice (Doo-Yeol Biotech Co. Ltd. Korea) Peritoneal macrophages were incubated with 10 μM SOCS3 recombinant proteins (1:HS3, 2:HM₁₆₅S3, 3:HM₁₆₅S3A and 4:HM₁₆₅S3B, respectively) for 1 hr at 37° C. and then stimulated them with LPS (Lipopolysaccharide)(500 ng/ml) and/or IFN-γ (100 U/ml) without removing iCP-SOCS3 proteins for 3, 6, or 9 hrs. The culture media were collected, and the extracellular levels of cytokine (TNF-α, IL-6) were measured by a cytometric bead array (BD Pharmingen, San Diego, Calif., USA) according to the manufacturer's instructions.

The effect of cell-permeable SOCS3 proteins on cytokines secretion was investigated. Treatment of C3H/HeJ primary peritoneal macrophages with SOCS3 proteins containing aMTD₁₆₅ suppressed TNF-α and IL-6 secretion induced by the combination of IFN-γ and LPS by 50-90% during subsequent 9 hrs of incubation (FIG. 24). In particular, aMTD₁₆₅/SDB-fused SOCS3 recombinant protein showed the greatest inhibitory effect on cytokine secretion. In contrast, cytokine secretion in macrophages treated with non-permeable SOCS3 protein (HS3) was unchanged, indicating that recombinant SOCS3 lacking the aMTD doesn't affect intracellular signaling. Therefore, we conclude that differences in the biological activities of HM₁₆₅S3B as compared to HS3B are due to the differences in protein uptake mediated by the aMTD sequence. In light of solubility/yield, cell-/tissue-permeability, and biological effect, SOCS3 recombinant protein containing aMTD and SDB (HM₁₆₅S3B) is a prototype of a new generation of improved cell-permeable SOCS3 (iCP-SOCS3), and will be selected for further evaluation as a potential anti-tumor agent.

Example 9. Preparation of Control Protein (Non-CP-SOCS3: HS3B Recombinant Protein)

As an experimental negative control, a SOCS3 recombinant protein having no cell permeability was prepared.

According to example 6-2, SOCS3 recombinant proteins lacking SD (HM₁₆₅S3) or both aMTD and SD (HS3) were found to be less soluble, produced lower yields, and showed tendency to precipitate when they were expressed and purified in E. coli. Therefore, we additionally designed and constructed SOCS3 recombinant protein containing only SDB (without aMTD₁₆₅: HS3B) as a negative control (FIG. 25). Preparation, expression and purification, and measurement of solubility/yield of the recombinant proteins were performed in the same manner as in Examples 6-2 and 6-3.

TABLE 37 Recombinant Cargo SD Protein 5′ Primers 3′ Primers SOCS3 B HS3B 5′-GGAATTC 5′-CGCGTCG CATATGGTCA ACTTAAAGGG CCCACAGCAA TTTCCGAAGG GTTTCCCGCC CTTGGCTATC GCC-3′ TT-3′

As expected, its solubility and yield increased compared to that of SOCS3 proteins lacking SDB (HS3; FIG. 26). Therefore, HS3B proteins were used as a control protein.

Example 10. Selection of aMTD for Cell-Permeability

After a basic structure of the stable recombinant proteins fused with combinations of aMTD and SD was determined, 22 aMTDs were selected for development of iCP-SOCS3 recombinant protein (Tables 38 and 39), based on the critical Factors, in order to examine which aMTD provides the highest cell-/tissue-permeability.

For comparison, 5 kinds of random peptides that do not satisfying one or more critical factors were selected (Table 40).

Solubility/yield and cell-permeability of 22 kinds of aMTD/SDB-fused SOCS3 recombinant proteins, prepared by using primers of Table 41 in the same manner as in Example 6-2, were analyzed according to examples 6-3 and 7-1, respectively.

TABLE 41  Amino Acid Cargo aMTD ID sequence 5′ Primers Primers SOCS3 MTM AAVLLPVLLAAP GGAATTCATATGGCGGCGGTGCTGC CGCGTCG TGCCGGTGCTGCTGGCGGCGCCGGT ACTTAAA CACCCAGAGCAAGTTTCCCGCCGCC GGGTTTC 44 ALAVPVALLVAP GGAATTCCATATGGCGCTGGCGGTGCC CGAAGGC GGTGGCGCTGCTGGTGGCGCCGGTCA TTGGCTA CCCACAGCAAGTTTCCCGCCGCC TCTT 81 AALLPALAALLP CGAATTCCATATGGCGGCGCTGCTGCC GGCGCTGGCGGCGCTGCTGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 123 AAIIVPAALLAP GGAATTCCATATGGCGGCGAATTGTG CCGGCGGCGCTGCTGGCGCCGGTCAC CCACAGCAAGTTTCCCGCCGCC 162 AVVALPAALIVP GGAATTCCATATGGCGGTGGTGGCGCT GCCGGCGGCGCTGATTGTGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 281 ALIVLPAAVAVP GGAATTCCATATGGCGCTGATTGTGCT GCCGGCGGCGGTGGCGGTGCCGGTC ACCCACAGCAAGTTTCCCGCCGCC 324 IVAVALPAALVP GGAATTCCATATGATTGTGGCGGTGGC GCTGCCGGCGGCGCTGGTGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 364 LVAAVAPALIVP GGAATTCCATATGCTGGTGGCGGCGGT GGCGCCGGCGCTGATTGTGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 365 AVIVVAPALLAP GGAATTCCATATGGCGGTGATTGTGGT GGCGCCGGCGCTGCTGGCGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 622 ALIVLAAPVAVP GGAATTCCATATGGCGCTGATTGTGCT GGCGGCGCCGGTGGCGGTGCCGGTC ACCCACAGCAAGTTTCCCGCCGCC 662 ALAVILAPVAVP GGAATTCCATATGGCGCTGGCGGTGAT TCTGGCGCCGGTGGCGGTGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 563 ALAVIVVPALAP GGAATTCCATATGGCGCTGGCGGTGAT TGTGGTGCCGGCGCTGGCGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 889 AVVIALPAVVAP GGAATTCCATATGGCGGTGGTGATTGC GCTGCCGGCGGTGGTGGCGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 897 AVIVPVAIIAAP GGAATTCCATATGGCGGTGATTGTGCC GGTGGCGATTATTGCGGCGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 623 VAAAIALPAIVP GGAATTCCATATGGTGGCGGCGGCGAT TGCGCTGCCGGCGATTGTGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 908 VALALAPVVVAP  GGAATTCCATATGGTGGCGCTGGCGCT GGCGCCGGTGGTGGTGGCGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 911 VALALPAVVVAP GGAATTCCATATGGTGGCGCTGGCGCT GCCGGCGGTGGTGGTGGCGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 2 AAAVPLLAVVVP GGAATTCCATATGGCGGCGGCGGTGC CGCTGCTGGCGGTGGTGGTGCCGGTC ACCCACAGCAAGTTTCCCGCCGCC 904 AVLAVVAPVVAP GGAATTCCATATGGCGGTGCTGGCGGT GGTGGCGCCGGTGGTGGCGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 481 AIAIAIVPVALP GGAATTCCATATGGCGATTGCGATTGC GATTGTGCCGGTGGCGCTGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 787 AVALVPVIVAAP  GGAATTCCATATGGCGGTGGCGCTGGT GCCGGTGATTGTGGCGGCGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 264 LAAAPVVIVIAP GGAATTCCATATGCTGGCGGCGGCGC CGGTGGTGATTGTGATTGCGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 363 AVLAVAPALIVP GGAATTCCATATGGCGGTGCTGGCGGT GGCGCCGGCGCTGATTGTGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 121 AIVALPALALAP GGAATTCCATATGGCGATTGTGGCGCT GCCGGCGCTGGCGCTGGCGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 921 IWWFVVLPLVVP GGAATTCCATATGATTTGGTGGTTTGTG GTGCTGCCGCTGGTGGTGCCGGTCAC CCACAGCAAGTTTCCCGCCGCC 16 NNSCTTYTNGSQ GGAATTCCATATGAACAACAGCTGCAC CACCTATACCAACGGCAGCCAGGTCA CCCACAGCAAGTTTCCCGCCGCC 67 LDAEVPLADDVP GGAATTCCATATGCTGGATGCGGAAGT GCCGCTGGCGGATGATGTGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 29 VLPPLPVLPVLP GGAATTCCATATGGTGCTGCCGCCGCT GCCGGTGCTGCCGGTGCTGCCGGTCA CCCACAGCAAGTTTCCCGCCGCC 700 GTSNTCQSNQNS GGAATTCCATATGGGCACCAGCAACAC CTGCCAGAGCAACCAGAACAGCGTCA CCCACAGCAAGTTTCCCGCCGCC

As shown in FIGS. 27 to 34, it was confirmed that most of the aMTD/SDB-fused SOCS3 recombinant proteins showed high solubility and yield and high cell permeability by aMTD. However, Random peptide-SOCS3-SDB recombinant protein showed remarkably low cell permeability.

Example 11-1. Investigation of Biological Activity for Determination of Optimal aMTD-Fused SOCS3 Recombinant Protein-1

Four kinds of aMTD/SD-fused SOCS3 recombinant proteins having high cell permeability and one kind of aMTD/SD-fused SOCS3 recombinant protein having the lowest cell permeability were selected, and their biological activity was analyzed.

PANC-1 cells (pancreatic carcinoma cell line) were seeded in a 16-well chamber slide at a density of 5×10³ cells/well, and then treated with 10 uM of aMTD/SD-fused SOCS3 for 24 hours. Apoptotic cells were analyzed using terminal dUTP nick-end labeling (TUNEL) assay with In Situ Cell Death Detection kit TMR red (Roche, 4056 Basel, Switzerland). Cells were treated with either 10 μM SOCS3 recombinant protein or buffer alone for 16 hrs with 2% fetal bovine serum (Hyclone, Logan, Utah, USA). Treated cells were washed with cold PBS two times, fixed in 4% paraformaldehyde (PFA, Junsei, Tokyo, Japan) for 1 hr at room temperature, and incubated in 0.1% Triton X-100 for 2 min on the ice. Cells were washed with cold PBS twice, and treated TUNEL reaction mixture for 1 hr at 37° C. in dark, washed cold PBS three times and observed by fluorescence microscopy (Nikon, Tokyo, Japan).

As shown in FIG. 35, most of the aMTD/SDB-fused SOCS3 recombinant proteins induced cell death of pancreatic carcinoma cells, and of them, aMTD₁₆₅ or aMTD₃₂₄-fused SOCS3 recombinant protein induced death of the largest number of cancer cells.

Example 11-2. Investigation of Biological Activity for Determination of Optimal aMTD-Fused SOCS3 Recombinant Protein-2

AGS cells (gastric carcinoma cell line) (American Type Culture Collection; ATCC) were seeded in a 12-well plate at a density of 1×10⁵ cells/well, and then treated with 10 uM of aMTD/SD-fused SOCS3 for 14 hours. Cancer cell death was analyzed by Annexin V analysis. Annexin V/7-Aminoactinomycin D (7-AAD) staining was performed using flow cytometry according to the manufacturer's guidelines (BD Pharmingen, San Diego, Calif., USA). Briefly, cells were washed three times with ice-cold PBS. The cells were then resuspended in 100 μl of binding buffer and incubated with 1 μl of 7-AAD and 1 μl of annexin V-PE for 30 min in the dark at 37° C. Flow cytometric analysis was immediately performed using a guava easyCyte™ 8 Instrument (Merck Millipore, Darmstadt, Germany).

As shown in FIG. 36, most of the aMTD/SDB-fused SOCS3 recombinant proteins induced cell death of gastric carcinoma cells, and of them, aMTD₁₆₅ or aMTD₂₈₁-fused SOCS3 recombinant protein induced death of the largest number of cancer cells.

Example 11-3. Investigation of Biological Activity for Determination of Optimal aMTD-Fused SOCS3 Recombinant Protein-3

AGS cells (gastric cancer cell line) were seeded in a 12 well plate at a density of 2.5×10⁵ per well, grown to 90% confluence. Confluent AGS cells were incubated with 10 μM HM#S3B in serum-free medium for 2 hrs prior to changing the growth medium (DMEM/F12, Hyclone, Logan, Utah, USA) and washed twice with PBS, and the monolayer at the center of the well was “wounded” by scraping with a sterilized white pipette tip. Cells were cultured for an additional 24 hrs and cell migration was observed by phase contrast microscopy (Nikon, ECLIPSE Ts2). The migration was quantified by counting the number of cells that migrated from the wound edge into the clear area.

As shown in FIG. 37, most of the aMTD/SDB-fused SOCS3 recombinant proteins inhibited cell migration of gastric carcinoma cells, and of them, aMTD₁₆₅ or aMTD₉₀₄-fused SOCS3 recombinant protein showed the most effective inhibition of cancer cell migration.

Solubility/yield, permeability, and biological activity of 22 kinds of the aMTD-fused recombinant proteins were examined in Examples 10 to 11-3, and as a result, the aMTD₁₆₅/SDB-fused SOCS3 recombinant protein was found to show the most excellent effect (FIG. 38). Therefore, the aMTD₁₆₅-fused recombinant protein was used in the subsequent experiment.

Example 12-1. Preparation of iCP-SOCS3 Recombinant Protein and Investigation of Equivalence Thereof-1

To develop a new drug as an anticancer agent, His-tag-removed iCP-SOCS3 recombinant protein was prepared and equivalence of His-Tag+, iCP-SOCS3 was investigated.

Histidine-tag free human SOCS3 proteins were constructed by amplifying the SOCS3 cDNA (225 amino acids) for aMTD fused to SOCS3 cargo. The PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, 1× reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctor protein, Korea)) were digested on the restriction enzyme site between Nde I (5′) and Sal I (3′) involving 35 cycles of denaturing (95° C.), annealing (62° C.), and extending (72° C.) for 45 sec each. For the last extension cycle, the PCR reactions remained for 10 min at 72° C. The PCR products were subcloned into pET-26b(+) (Novagen, Darmstadt, Germany). Coding sequence for SDB fused to C terminus of aMTD-SOCS3 was cloned at BamHI (5′) and SalI (3′) in pET-26b(+) from PCR-amplified DNA segments and confirmed by DNA sequence analysis of the resulting plasmids.

TABLE 42 Recombinant Cargo SD Protein 5′ Primers 3′ Primers SOCS3 — HS3 5′-GGAATTC 5′-CCCGGAT CATATGGTCA CCTTAAAGCG CCCACAGCAA GGGCATCGTA GTTTCCCGCC CTGGTCCAGG GCC-3′ AA-3′ — HM₁₆₅S3 5′-GGAATTC 5′-CCCGGAT CATATGGCGC CCTTAAAGCG TGGCGGTGCC GGGCATCGTA GGTGGCGCTG CTGGTCCAGG GCGATTGTGC AA-3′ CGGTCACCCA CAGCAAGTTT C-3′ A HM₁₆₅S3A 5′-GAATTCC 5′-CGCGTCG ATATGGCGCT ACTTACCTCG GGCGGTGCCG GCTGCACCGG GTGGCGCTGG CACGGCGATA CGATTGTGCC C-3′ GGTCACCCAC AGCAAGTTT C-3′ B HM₁₆₅S3B 5′-GGAATTC 5′-CGCGTCG CATATGGCGC ACTTAAAGGG TGGCGGTGCC TTTCCGAAGG GGTGGCGCTG CTTGGCTATC GCGATTGTGC TT-3′ CGGTCACCCA CAGCAAGTTT C-3′ C HM₁₆₅S3C 5′-GGAATTC 5′-GCGTCGA CATATGGCGC CTTAGGCCAG TGGCGGTGCC GTTAGCGTCG GGTGGCGCTG AG-3′ GCGATTGTGC CGGTCACCCA CAGCAAGTTT C-3′ D HM₁₆₅S3D 5′-GGAATTC 5′-GCGTCGA CATATGGCGC CTTATTTTTT TGGCGGTGCC CTCGGACAGA GGTGGCGCTG TA-3′ GCGATTGTGC CGGTCACCCA CAGCAAGTTT C-3′ E HM₁₆₅S3E 5′-GGAATTC 5′-ACGCGTC CATATGGCGC GACTTAACCT TGGCGGTGCC CCAATCTGTT GGTGGCGCTG CGCGGTGAGC GCGATTGTGC CTC-3′ CGGTCACCCA CAGCAAGTTT C-3′ SOCS3 B M₁₆₅S3B 5′-GGAATTC 5′-CGCGTCG CATATGOCGC ACTTAAAGGG TGGCGGTGCC TTTCCGAAGG GGTGGCGCTG CTTGGCTATC GCGATTGTGC TT-3′ CGGTCACCCA CAGCAAGTTT C-3′

Expression, purification and solubility/yield were measured in the same manner as in Examples 6-2 and 6-3, and as a result, his-tag-removed M₁₆₅S3B was found to have high solubility/yield (FIG. 39).

Example 12-2. Preparation of iCP-SOCS3 Recombinant Protein and Investigation of Equivalence Thereof-2

In the same manner as in Example 7-1, RAW264.7 cells were treated with FITC-labeled HS3B, HM₁₆₅S3B, and M₁₆₅S3B proteins, and cell permeability was evaluated.

As shown in FIG. 40, both HM₁₆₅S3B and M₁₆₅S3B were found to have high cell permeability.

Example 12-3. Preparation of iCP-SOCS3 Recombinant Protein and Investigation of Equivalence Thereof-3

To investigate biological activity equivalence of the HM₁₆₅S3B and M₁₆₅S3B recombinant proteins, induction of apoptosis of gastric carcinoma cell line (AGS) was analyzed by Annexin V staining in the same manner as in Example 11-2, and inhibition of migration was analyzed in the same manner as in Example 11-3.

As shown in FIG. 41, it was confirmed that both HM₁₆₅S3B and M₁₆₅S3B showed high anticancer efficacy and M₁₆₅S3B exhibited efficacy equivalent to or higher than HM₁₆₅S3B.

Example 12-4. Preparation of iCP-SOCS3 Recombinant Protein and Investigation of Equivalence Thereof-4

In silico MHC class II binding analysis using iTope™ (ANTITOPE.LTD) revealed changing the V28 p1 anchor residue in SDB sequence to L makes this region human germline and as such both MHC class II binding peptides within this region would be expected to be low risk due to T cell tolerance.

To prepare humanized SDB domain, iCP-SOCS3 was prepared as in FIG. 50. iCP-SOCS3 was prepared in the same manner as in Example 6-1, except that SDB′ having a substitution of valine with leucine at an amino acid position 28 was used (FIG. 78). Further, protein purification was performed in the same manner as in example 6-2 using the primer as below (Table 43).

TABLE 43 Recombinant Cargo SD Protein 5′ Primers 3′ Primers SOCS3 — HS3 5′-GGAATTC 5′-CCCGGAT CATATGGTCA CCTTAAAGCG CCCACAGCAA GGGCATCGTA GTTTCCCGCC CTGGTCCAGG GCC-3′ AA-3′ — HM₁₆₅S3 5′-GGAATTC 5′-CCCGGAT CATATGGCGC CCTTAAAGCG TGGCGGTGCC GGGCATCGTA GGTGGCGCTG CTGGTCCAGG GCGATTGTGC AA-3′ CGGTCACCCA CAGCAAGTTT C-3′ A HM₁₆₅S3A 5′-GGAATTC 5′-CGCGTCG CATATGGCGC ACTTACCTCG TGGCGGTGCC GCTGCACCGG GGTGGCGCTG CACGGCGATA GCGATTGTGC C-3′ CGGTCACCCA CAGCAAGTTT C-3′ B HM₁₆₅S3B 5′-GGAATTC 5′-CGCGTCG CATATGGCGC ACTTAAAGGG TGGCGGTGCC TTTCCGAAGG GGTGGCGCTG CTTGGCTATC GCGATTGTGC TT-3′ CGGTCACCCA CAGCAAGTTT C-3′ C HM₁₆₅S3C 5′-GGAATTC 5′-GCGTCGA CATATGGCGC CTTAGGCCAG TGGCGGTGCC GTTAGCGTCG GGTGGCGCTG AG-3′ GCGATTGTGC CGGTCACCCA CAGCAAGTTT C-3′ D HM₁₆₅S3D 5′-GGAATTC 5′-GCGTCGA CATATGGCGC CTTATTTTTT TGGCGGTGCC CTCGGACAGA GGTGGCGCTG TA-3′ GCGATTGTGC CGGTCACCCA CAGCAAGTTT C-3′ E HM₁₆₅S3E 5′-GGAATTC 5′-ACGCGTC CATATGGCGC GACTTAACCT TGGCGGTGCC CCAATCTGTT GGTGGCGCTG CGCGGTGAGC GCGATTGTGC CTC-3′ CGGTCACCCA CAGCAAGTTT C-3′ SOCS3 B* HM₁₆₅S3B* 5′-GGAATTC 5′-CGCGTCG CATATGGCGC ACTTAAAGGG TGGCGGTGCC TTTCCGAAGG GGTGGCGCTG CTTGGCTATC GCGATTGTGC TT-3′ CGGTCACCCA CAGCAAGTTT C-3′

As shown in FIG. 44, both HM₁₆₅S3B and HM₁₆₅S3B′(V28L) were found to have high solubility/yield.

Example 12-5. Preparation of iCP-SOCS3 Recombinant Protein and Investigation of Equivalence Thereof-5

In the same manner as in Example 7-1, RAW264.7 cells were treated with FITC-labeled HM₁₆₅S3B and HM₁₆₅S3B′(V28L) proteins, and cell permeability was evaluated.

As shown in FIG. 45, both HM₁₆₅S3B and HM₁₆₅S3B′(V28L) were found to have high cell permeability.

Example 12-6. Preparation of iCP-SOCS3 Recombinant Protein and Investigation of Equivalence Thereof-6

To investigate biological activity equivalence of the HM₁₆₅S3B and HM₁₆₅S3B′(V28L) recombinant proteins, anti-proliferative activity was examined and induction of apoptosis of gastric carcinoma cell line (AGS) was analyzed by Annexin V staining in the same manner as in Example 11-2, and inhibition of migration was analyzed in the same manner as in Example 11-3.

Antiproliferative activity were evaluated with the CellTiter-Glo Cell Viability Assay. AGS cells (3×10³/well) were seeded in 96 well plates. The next day, cells were treated with DMEM (vehicle) or 10 μM HM₁₆₅S3B, HM₁₆₅S3B′(V28L) for 96 hrs in the presence of serum (2%). Proteins were replaced daily. Cell growth and survival were evaluated with the CellTiter-Glo Cell Viability Assay (Promega, Madison, Wis.). Measurements using a Luminometer (Turner Designs, Sunnyvale, Calif.) were conducted following the manufacturer's protocol.

It was confirmed that both HM₁₆₅S3B and HM₁₆₅S3B′(V28L) showed high anti-proliferative effects on gastric carcinoma cells (FIG. 46), and also effects of inducing apoptosis (FIG. 47) and of inhibiting migration of gastric carcinoma cells (FIG. 48), and in particular, HM₁₆₅S3B′(V28L) exhibited efficacy equivalent to or higher than HM₁₆₅S3B.

Example 12-7. Preparation of iCP-SOCS3 Recombinant Protein and Investigation of Equivalence Thereof-7

iCP-SOCS3 of BS3M₁₆₅ structure was prepared in the same manner as in Example 6-1, and B′S3M₁₆₅ iCP-SOCS3 was also prepared by humanized SDB domain (FIG. 49a ).

TABLE 44 Recombinant cargo SD Protein 5′ Primers 3′ Primers SOCS3 B BS3M₁₆₅ 5′-GGAATTC 5′-ACGCGTC CATATGATGG GACTTACGCC CAGAACAAAG AGCGCCACCG CGAC-3′ GCACCGCCAG CGCAATCACC GGAAGCGGGG CATCGTACTG GTCCAG-3′ B* B*S3M₁₆₅ 5′-GGAATTC 5′-AGGCGTC CATATGATGG GACTTACGCC CAGAACAAAG AGCGCCACCG CGAC-3′ GCACCGCCAG CGCAATCACC GGAAGCGGGG CATCGTACTG GTCCAG-3′

Expressions and purifications of iCP-SOCS3 recombinant protein (BS3M₁₆₅, B′S3M₁₆₅) in E. coli (bottom) were analyzed in the same manner as in Examples 6-2 and 6-3, respectively, and were shown in FIG. 49b . Further, E. coli codon-optimized iCP-SOCS3 was prepared.

Example 13. Test of Biological Activity of iCP-SOCS3—Inhibition Activity of IFN-γ-Induced STAT Phosphorylation

Whether iCP-SOCS3 (HM₁₆₅S3B) recombinant protein inhibits activation of the JAK/STAT-signaling pathway was examined by the method of Example 8-1.

PANC-1 Cells (KCLB, Seoul, South Korea) were treated with 10 ng/ml IFN-γ (R&D systems, Abingdon, UK) for 15 min and treated with either DMEM (vehicle) or 1, 5, 10 μM aMTD/SD-fused SOCS3 recombinant proteins for 2 hrs. The cells were lysed in RIPA lysis buffer (Biosesang, Seongnam, Korea) containing proteinase inhibitor cocktail (Roche, Indianapolis, Ind., USA), incubated for 15 min at 4° C., and centrifuged at 13,000 rpm for 10 min at 4° C. Equal amounts of lysates were separated on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. The membranes were blocked using 5% skim milk in TBST and for western blot analysis incubated with the following antibodies: anti-phospho-STAT3 (Cell Signaling Technology, USA), then HRP conjugated anti-rabbit secondary antibody (Santacruz).

The well-known cytokine signaling inhibitory actions of SOCS3 are inflammation inhibition through i) inhibition of IFN-γ-mediated JAK/STAT and ii) inhibition of LPS-mediated cytokine secretion. The ultimate test of cell-penetrating efficiency is a determination of intracellular activity of SOCS3 proteins transported by aMTD. Since endogenous SOCS3 are known to block phosphorylation of STAT3 by IFN-γ-mediated Janus kinases (JAK) 1 and 2 activation, we demonstrated whether cell-permeable SOCS3 inhibits the phosphorylation of STATs. As shown in FIG. 50, iCP-SOCS3 (HM₁₆₅S3B) suppressed IFN-γ-induced phosphorylation of STAT3 in dose dependent manner. In contrast, STAT phosphorylation was readily detected in cells exposed to HS3B, which lacks the aMTD motif required for membrane penetration, indicating that HS3B, which lacks an MTD sequence did not enter the cells, has no biological activity.

Example 14. Investigation of Basic CPP-Mediated Intracellular Delivery Mechanism

The mechanism of basic CPP (TAT and PolyR)-mediated intracellular delivery was also investigated in the same manner as in Example 7-1 and Example 14-2.

As shown in FIG. 83, it was confirmed that aMTD165/SD-fused SOCS3 recombinant proteins are independent to cell surface receptor (FIG. 83A) and the cell-permeability of aMTD165/SD-fused SOCS3 recombinant proteins is not due to endocytosis (FIG. 83B).

Whether cells treated with aMTD165/SD-fused SOCS3, TAT/SD-fused SOCS3, and PolyR/SD-fused SOCS3 could transfer the protein to neighboring cells were also tested on a molecular level in the same manner as in Example 13.

For this, RAW 264.7 cells were treated with 5 μM of FITC-labeled HM₁₆₅S3B, HTS3B for 2 hour and washed with PBS two times. Thereafter, they were seeded on PANC-1 cell, incubated for 2 hours and treated with 20 ng/ml of IFN-γ for 15 minutes, followed by Western blotting in the same manner as in Example 8-1. And Cell-to-cell protein transfer was assessed by flow cytometry.

As shown in FIG. 84, efficient cell-to-cell transfer of HM₁₆₅S3B, but not HTS3B or HRS3B, suggests that only SOCS3 recombinant proteins containing aMTD165 are capable of bidirectional passage across the plasma membrane.

Moreover, as shown in FIG. 85, phospho-STAT3 was only reduced in cells treated with HM₁₆₅S3B.

Example 14-1. Investigation of Time- and Dose-Dependency of iCP-SOCS3 Cell-Permeability

It was examined whether the cell-permeability of iCP-SOCS3 recombinant protein is dose-dependent. Cell-permeability of iCP-SOCS3 recombinant protein was tested in the same manner as in Example 7-1 except for the cells being treated with 0.05 μM˜10 μM of iCP-SOCS3 recombinant protein for 1 hrs. As shown in FIGS. 86a and 86b , it was confirmed that the cell-permeability of iCP-SOCS3 recombinant protein is dose-dependent

Time-dependency of cell-permeability was also investigated. The cells were treated with 10 μM of iCP-SOCS3 recombinant protein for 5˜180 minutes. As shown in FIG. 87, high level of cell-permeability of iCP-SOCS3 was observed only 5 minutes post-treatment and could be seen even at 180 minutes mark.

Example 14-2. Investigation of aMTD-Mediated Intracellular Delivery Mechanism

The mechanism of aMTD₁₆₅-mediated intracellular delivery was investigated.

(1) RAW 264.7 cells were pretreated with 100 mM EDTA for 3 hours, and then treated with 10 uM of iCP-SOCS3 (HM₁₆₅S3B) protein for 1 hour, followed by flow cytometry in the same manner as in Example 7-1 (FIG. 51A).

(2) RAW 264.7 cells were pretreated with 5 ug/ml of proteinase K for 10 minutes, and then treated with 10 uM of iCP-SOCS3 (HM₁₆₅S3B) protein for 1 hour, followed by flow cytometry (FIG. 51B).

(3) RAW 264.7 cells were pretreated with 20 uM taxol for 30 minutes, and then treated with 10 uM of iCP-SOCS3 (HM₁₆₅S3B) protein for 1 hour, followed by flow cytometry (FIG. 52A).

(4) RAW 264.7 cells were pretreated with 1 mM ATP and 10 uM antimycin singly or in combination for 2 hours, and then treated with 10 uM of iCP-SOCS3 (HM₁₆₅S3B) protein for 1 hour, followed by flow cytometry (FIG. 52B).

(5) RAW 264.7 cells were left at 4° C. and 37° C. for 1 hour, respectively, and then treated with 10 uM of iCP-SOCS3 (HM₁₆₅S3B) protein for 1 hour, followed by flow cytometry (FIG. 53).

The aMTD-mediated intracellular delivery of SOCS3 protein did not require protease-sensitive protein domains displayed on the cell surface (FIG. 51B), microtubule function (FIG. 52A), or ATP utilization (FIG. 52B), since aMTD₁₆₅-dependent uptake, compare to HS3 and HS3B, was essentially unaffected by treating cells with proteinase K, taxol, or the ATP depleting agent, antimycin. Conversely, iCP-SOCS3 (HM₁₆₅S3B) proteins uptake was blocked by treatment with EDTA and low temperature (FIGS. 51A and 53), indicating the importance of membrane integrity and fluidity for aMTD-mediated protein transduction.

Moreover, whether cells treated with iCP-SOCS3 (HM₁₆₅S3B) protein could transfer the protein to neighboring cells were also tested.

For this, RAW 264.7 cells were treated with 10 uM of FITC-labeled iCP-SOCS3 (HM₁₆₅S3B) protein for 1 hour. Thereafter, these cells were co-cultured with PerCP-Cy5.5-CD14-stained RAW 264.7 cells for 2 hours. Cell-to-cell protein transfer was assessed by flow cytometry, scoring for CD14/FITC double-positive cells. Efficient cell-to-cell transfer of HM₁₆₅S3B, but not HS3 or HS3B (FIG. 54), suggests that SOCS3 recombinant proteins containing aMTD₁₆₅ are capable of bidirectional passage across the plasma membrane.

Example 15. Investigation of Bioavailability of iCP-SOCS3

To investigate BA of the iCP-SOCS3 (HM₁₆₅S3B) recombinant proteins, ICR mice (Doo-Yeol Biotech Co. Ltd., Seoul, Korea) were intravenous (IV) injected with 600 μg/head of 10 μM FITC (Fluorescein isothiocyanate, SIGMA, USA)-labeled SOCS3 recombinant proteins (HS3B, HM₁₆₅S3B) and after 15 min, 30 min, 1H, 2H, 4H, 8H, 12H, 16H, 24H, 36H, 48H, mice of each group were sacrificed. From the mice, peripheral blood mononuclear cells (PBMCs), splenocytes, and hepatocytes were separated.

Further, the spleen was removed and washed with PBS, and then placed on a dry ice and embedded in an O.C.T. compound (Sakura). After cryosectioning at 20 μm, tissue distributions of fluorescence-labeled-SOCS3 proteins in different organs was analyzed by fluorescence microscopy (Carl Zeiss, Gottingen, Germany).

Isolation of PBMC

After anesthesia with ether, ophthalmectomy was performed and the blood was collected therefrom using a 1 ml syringe. The collected blood was immediately put in an EDTA tube and mixed well. The blood was centrifuged at 4,000 rpm and 4° C. for 5 minutes, and plasma was discarded and only buffy coat was collected in a new microtube. 0.5 ml of RBC lysis buffer (Sigma) was added thereto, followed by vortexing. The microtube was left at room temperature for 5 minutes, and then centrifuged at 4,000 rpm and 4° C. for 5 minutes. 0.3 ml of PBS was added to a pellet, followed by pipetting and flow cytometry (FlowJo cytometric analysis software, Guava, Millipore, Darmstadt, Germany).

Isolation of Splenocytes and Hepatocytes

Mice were laparotomized and the spleen or liver were removed. The spleen or liver thus removed was separated into single cells using a Cell Strainer (SPL, Korea). These cells were collected in a microtube, followed by centrifugation at 4,000 rpm and 4° C. for 5 minutes. 0.5 ml of RBC lysis buffer was added thereto, followed by vortexing. The microtube was left at room temperature for 5 minutes, and then centrifuged at 4,000 rpm and 4° C. for 5 minutes. 0.5 ml of PBS was added to a pellet, followed by pipetting and flow cytometry (FlowJo cytometric analysis software, Guava, Millipore, Darmstadt, Germany).

As shown in FIG. 55, in PBMCs, the maximum permeability of iCP-SOCS3 was observed at 30 minutes, and in splenocytes, the maximum permeability of iCP-SOCS3 was observed at 2 hours and maintained up to 16 hours. In hepatocytes, the maximum permeability of iCP-SOCS3 was observed at 15 minutes and maintained up to 16 hours. As shown in FIG. 56, in the pancreas tissue, very high distribution of iCP-SOCS3 was observed at 2 hours, and maintained up to 8 hours. Therefore, it can be seen that iCP-SOCS3 is rapidly delivered from blood to various tissues within 2 hours, and maintained up to 8˜16 hours depending on the tissues.

Example 16. Investigation of Anticancer Efficacy of iCP-SOCS3 Recombinant Protein

To develop the iCP-SOCS3 recombinant protein as a therapeutic agent for hepatocellular carcinoma, its permeability to hepatocellular carcinoma cell lines and liver tissues was investigated.

HepG2 cells (ATCC, Manassas, Va., USA) were seeded in a 12 well plate, grown to 90% confluence. Cells were treated with 10 μM FITC-labeled iCP-SOCS3 recombinant proteins and cultivated for 1 hr at 37° C.

After cultivation, the cells were treated with proteinase K (10 μg/mL, SIGMA, USA) and washed three times with ice-cold PBS (Phosphate-buffered saline, Hyclone, USA) to remove surface-bound proteins, and internalized proteins were measured by flow cytometry (FlowJo cytometric analysis software, Guava, Millipore, Darmstadt, Germany). Untreated cells (gray) and equimolar concentration of unconjugated FITC (FITC only, green)-treated cells were served as control (FIG. 57).

The liver tissue permeability was analyzed by the method described in Example 7-2.

As shown in FIG. 57, iCP-SOCS3 recombinant proteins (FITC-HM₁₆₅S3SB) promoted the transduction into cultured HCC HepG2 cells. In contrast, SOCS3 proteins containing non-aMTD (FITC-HS3 and FITC-HS3B) did not appear to enter cells.

In addition, iCP-SOCS3 recombinant proteins (FITC-HM₁₆₅S3SB) enhanced the systemic delivery to liver after intraperitoneal injection (FIG. 58). Therefore, these data indicate that iCP-SOCS3 protein could be intracellularly delivered and distributed to the hepatocytes and liver tissue, contributing for beneficial biotherapeutic effects.

Example 16-1. Investigation of Tumor Targeting of iCP-SOCS3

Tumor-targeting of the anticancer agent iCP-SOCS3 (HM₁₆₅S3B) recombinant proteins was investigated in an orthotopic xenograft model. The protocol was adapted from World J Gastroenterol. 2014 Jul. 28; 20(28): 9476-9485. To generate an orthotopic xenograft mouse model, PANC-1-Luc cells (1×10⁶ cells/mouse) cultured in RPMI 1640 (Hyclone, Logan, Utah, USA) containing 10% FBS were mixed with Matrigel at a 1:1 ratio and 200 μl of the mixture were injected into pancreas tail of female Balb/c^(nu/nu) mice (5-week-old, Doo-Yeol Biotech Co. Ltd. Korea). After 7 weeks, the mice were IP injected with 100 μl of luciferin (Sigma-Aldrich, St. Louis, Mo., USA) and imaged by Xenogen IVIS-200 imaging system 15 minute later. As shown in FIG. 88 (A), pancreatic cancer model was well established.

The iCP-SOCS3 recombinant proteins were labeled and IV injected to the mice with 40 mg/kg according to a manual of Alexa680 kit (Thermo Fisher Scientific, San Jose, Calif., USA). As shown in FIG. 88 (B), the iCP-SOCS3 recombinant proteins were delivered to every part of the body at 5 minutes and more iCP-SOCS3 recombinant proteins were accumulated in pancreas. As time passed, a large amount of the proteins were accumulated only in tumor. Therefore, the iCP-SOCS3 recombinant protein was proved to be a mechanism-specific anticancer agent that specifically targets cancer tissues.

Example 17. Investigation of Anticancer Efficacy of iCP-SOCS3 Recombinant Protein

To develop the iCP-SOCS3 recombinant protein as a mechanism-specific therapeutic agent for hepatocellular carcinoma, SOCS3 levels endogenously expressed and activation of JAK/STAT-signaling pathway were investigated in different hepatocellular carcinoma cell lines, normal cells, and normal hepatic cells (ATCC, Manassas, Va., USA & KCLB, Seoul, South Korea).

Example 17-1. Analysis of Hypermethylation Level in Cell Line

SOCS3 expression is suppressed due to methylation in cancer cells, and therefore, inflammation or cancer development is increased. Further, to investigate the effect of cell permeable SOCS3, a cancer cell line where endogenous SOCS3 expression is suppressed should be selected or applied to a model, and therefore, the present experiments were performed.

Genomic DNA was extracted from cancer cell line using an Exgene™ Tissue SV mini kit (Geneall®, Korea). DNA was quantified, and experiments were performed using 500 ng of gDNA and an EZ DNA Methylation-Gold™ kit (ZYMO Research, Orange, Calif., USA) according to the manufacturer's instructions. DNA was used to perform PCR, and methylation and unmethylation of endogenous SOCS3 were qualitatively analyzed by electrophoresis. In this regard, the primers used are as follows.

Unmethyl-F was 5′-tag tgt gta agt tgt agg aga gtg g-3′(SEQ ID NO: 816), Unmethyl-R was 5′-cta aac ata aaa aaa taa cac taa tcc aaa-3′ (SEQ ID NO: 817), Methyl-F was 5′-gta gtg cgt aag ttg tag gag agc-3′ (SEQ ID NO: 818), and Methyl-R was 5′-gta aaa aaa taa cgc taa tcc gaa-3′ (SEQ ID NO: 819). PCR was performed for 30 cycles consisting of pre-denaturation at 95° C. for 5 minutes, denaturation at 95° C. for 30 seconds, annealing at 60° C. for 45 seconds, and extension at 72° C. for 1 minute, and then final extension at 72° C. for 8 minutes.

As shown in FIG. 59, unmethylation of SOCS3 was observed in HEK293 and Chang cells which are normal cells, whereas hypermethylation of the promoter region of SOCS3 gene was observed in HepG2, Hep3B, Huh-7, and SK-HEP-1 which are hepatocellular carcinoma cell lines (U: unmethylated SOCS3, M: methylated SOCS3). These results indicate that SOCS3 is silenced by hypermethylation in hepatocellular carcinoma cell lines.

Example 17-2. Analysis of Expression Level of Endogenous SOCS3 mRNA in Cell Line

SOCS3 mRNA expression levels in cancer cells were analyzed by RT-PCR. mRNAs were isolated from normal cell lines and cancer cell lines according to a method provided in a manufacturer's sheet of Hybrid-R (Geneall, Korea), and PCR was performed using SOCS3 primer F 5′-cct act gaa ccc tcc tcc ga-3′ (SEQ ID NO: 820) and SOCS3 primer R 5′-gca get ggg tga ctt tct ca-3′ (SEQ ID NO: 821) for 30 cycles consisting of denaturing (95° C.), annealing (60° C.), and extending (72° C.) for 45 seconds each.

The results of electrophoresis showed that high expression levels of SOCS3 were observed in the normal HEK293 and Chang cells whereas low expression levels of SOCS3 were observed in the hepatocellular carcinoma cell lines, HepG2, Hep3B, and Huh-7 (FIG. 60).

Example 17-3. Analysis of Expression of Endogenous SOCS3 and JAK/STAT Signaling Activation Status in HCC Cell Line

JAK/STAT3 activation in cancer cells was analyzed by Western blot analysis. The normal HEK293 and Chang cells, and hepatocellular carcinoma cell lines, HepG2, Hep3B, Huh-7, and SK-HEP-1 were washed with PBS, and then the cells were lysed in RIM lysis buffer (Biosesang, Seongnam, Korea) containing proteinase inhibitor cocktail (Roche, Indianapolis, Ind., USA), incubated for 15 min at 4° C., and centrifuged at 13,000 rpm for 10 min at 4° C. to isolate proteins, followed by western blotting in the same manner as in Example 8-1.

As shown in FIG. 61, low JAK1 and JAK2 phosphorylations were observed in normal HEK293 and Chang cells, whereas low expression levels of SOCS3 gene were observed in the hepatocellular carcinoma cell lines, HepG2, Hep3B, and Huh-7. These results suggest a possibility of developing a mechanism-specific anticancer agent, because SOCS3-deficient cancer cells can be replenished with cell permeable proteins, and activated JAK/STAT-signaling can be negatively regulated.

Example 18. Investigation of Anti-Cancer Efficacy (Anti-Proliferative Activity) of iCP-SOCS3

In order to develop the iCP-SOCS3 recombinant protein as a therapeutic agent for hepatocellular carcinoma, efficacy of iCP-SOCS3 on proliferation of hepatocellular carcinoma cell lines was investigated.

Cells originated from human hepatocellular carcinoma cell (Hep3B, HepG2, Huh-7, SK-Hep1), human normal cells (HEK293, HaCaT, Chang liver cell) and mouse fibroblast (NIH3T3) were purchased from ATCC (Manassas, Va., USA) and KCLB (Seoul, Korea). All the cells were maintained as recommended by the supplier. These cells (3×10³/well) were seeded in 96 well plates. The next day, cells were treated with DMEM (vehicle) or 10 μM recombinant SOCS3 proteins for 96 hrs in the presence of serum (2%). Proteins were replaced daily. Cell growth and survival were evaluated with the CellTiter-Glo Cell Viability Assay (Promega, Madison, Wis.). Measurements using a Luminometer (Turner Designs, Sunnyvale, Calif.) were conducted following the manufacturer's protocol.

Since the endogenous level of SOCS3 protein is reduced in hepatocellular carcinoma patient, and SOCS3 negatively regulates cell growth and motility in cultured HCC cells, we investigated whether iCP-SOCS3 inhibits cell viability through SOCS3 intracellular replacement in HCC cells. As shown in FIG. 62, SOCS3 recombinant proteins containing aMTD₁₆₅ significantly suppressed cancer cell proliferation. iCP-SOCS3 (HM₁₆₅S3B) protein was the most cytotoxic to Hep3B2.1-7 hepatocellular carcinoma cells—over 80% in 10 μM treatment (p<0.01)—especially compared to vehicle alone (i.e. exposure of cells to culture media without recombinant proteins; left). However, neither cell-permeable SOCS3 protein adversely affected the cell viability of non-cancer cells (Chang cell) even after exposing these cells to equal concentrations (10 μM) of protein over 4 days. These results suggest that the iCP-SOCS3 protein is not overly toxic to normal cells and selectively kills tumor cells, and would have a great ability to inhibit cell survival-associated phenotypes in hepatocellular carcinoma without any severe aberrant effects as a protein-based biotherapeutics.

Example 19. Investigation of Anti-Cancer Efficacy (Cell Migration Inhibition) of iCP-SOCS3

In order to develop the iCP-SOCS3 recombinant protein as a therapeutic agent for hepatocellular carcinoma, efficacy of iCP-SOCS3 on migration and metastasis of hepatocellular carcinoma cell lines were investigated.

Cells were seeded into 12-well plates, grown to 90% confluence, and incubated with 10 μM Non-CP-SOCS3 (HS3B), iCP-SOCS3 (HM₁₆₅S3B) in serum-free medium for 2 hrs prior to changing the growth medium (recommended medium by the supplier). The cells were washed twice with PBS, and the monolayer at the center of the well was “wounded” by scraping with a pipette tip. Cells were cultured for an additional 24˜72 hrs and cell migration was observed by phase contrast microscopy (Nikon, ECLIPSE Ts2). The migration was quantified by counting the number of cells that migrated from the wound edge into the clear area.

As shown in FIG. 63, iCP-SOCS3 (HM₁₆₅S3B) suppressed the repopulation of wounded monolayer although SOCS3 protein lacking aMTD₁₆₅ (HS3B) had no effect on the cell migration, in HepG2 and Huh-7 cells. In normal Chang liver cells, HM₁₆₅S3B protein (iCP-SOCS3) had no effect on the cell migration.

Transwell Migration Assay

The lower surface of Transwell inserts (Costar) was coated with 0.1% gelatin, and the membranes were allowed to dry for 1 hr at room temperature. The Transwell inserts were assembled into a 24-well plate, and the lower chamber was filled with growth media containing 10% FBS and FGF2 (40 ng/ml). Cells were added to each upper chamber at a density of 5×10⁵, and the plate was incubated at 37° C. in a 5% CO₂ incubator for 24 hrs. Migrated cells were stained with 0.6% hematoxylin and 0.5% eosin and counted.

As shown in FIGS. 64a to 64c , HepG2, Huh-7, SK-Hep-1 cells treated with iCP-SOCS3 (HM₁₆₅S3B) protein also showed significant inhibitory effect on their Transwell migration compared with untreated cells (Vehicle) and non-permeable SOCS3 protein-treated cells.

Invasion Assay

The lower surface of Transwell inserts (Costar) was coated with 0.1% gelatin, the upper surface of Transwell inserts was coated with matrigel (40 μg per well; BD Pharmingen, San Diego, Calif., USA), and the membranes were allowed to dry for 1 hr at room temperature. The Transwell inserts were assembled into a 24-well plate, and the lower chamber was filled with growth media containing 10% FBS and FGF2 (40 ng/ml). Cells (5×10⁵) were added to each upper chamber, and the plate was incubated at 37° C. in a 5% CO₂ incubator for 24 hrs. Migrated cells were stained with 0.6% hematoxylin and 0.5% eosin and counted.

As shown in FIG. 65, HepG2 cells treated with iCP-SOCS3 (HM₁₆₅S3B) protein caused remarkable decrease in invasion compared with the control proteins. Taken together, these data indicate that iCP-SOCS3 contributes to inhibit tumorigenic activities of hepatocellular carcinoma cells.

Example 20-1. Investigation of Anti-Cancer Efficacy (Induction of Apoptosis) of iCP-SOCS3

To further determine the effect of iCP-SOCS3 on the tumorigenicity of hepatocellular carcinoma cells, we subsequently investigated whether iCP-SOCS3 regulates apoptosis in HepG2 cells.

Annexin V/7-Aminoactinomycin D (7-AAD) staining was performed using flow cytometry according to the manufacturer's guidelines (BD Pharmingen, San Diego, Calif., USA). Briefly, 1×10⁶ cells were washed three times with ice-cold PBS. The cells were then resuspended in 100 μl of binding buffer and incubated with 1 μl of 7-AAD and 1 μl of Annexin V-7-AAD for 30 min in the dark at 4° C. Flow cytometric analysis was immediately performed using a guava easyCyte™ 8 Instrument (Merck Millipore, Darmstadt, Germany). In this regard, Cisplatin® (IC₅₀: 30 μM) was used as a reference drug.

As shown in FIG. 66, HM₁₆₅S3B protein (iCP-SOCS3) was a dose-dependently efficient inducer of apoptosis in HepG2 cells, as assessed by Annexin V staining. Further, upon treatment of 10 uM or more of iCP-SOCS3, there was no difference in induction of apoptosis. Consistently, no changes in Annexin V staining were observed in HepG2 cells treated with HS3B compared to untreated cell (Vehicle). However, the reference drug, Cisplatin® hardly induced apoptosis. Accordingly, the concentration of iCP-SOCS3 was determined as 10 uM.

Example 20-2. Investigation of Anti-Cancer Efficacy (Induction of Apoptosis) of iCP-SOCS3

The effect of inducing apoptosis of Chang liver cells was measured in the same manner as in Example 11-2, except that as target cell lines, normal Chang liver cells and hepatocellular carcinoma cell lines, Hep3B, HepG2, Huh7, and SK-HEP-1 were used, and as a reference drug, 5-FU® (IC₅₀: 40 μM) was used.

The iCP-SOCS3 (HM₁₆₅S3B) protein was a considerably efficient inducer of apoptosis in HCCs (Hep3B, HepG2, Huh7, and SK-HEP-1), as assessed by Annexin V staining (FIG. 68) while dose not induced apoptosis in normal cells (Chang-Liver) (FIG. 67). Consistently, no changes in Annexin V staining were observed in HepG2 cells treated with HS3B compared to untreated cell (Vehicle). Accordingly, it was confirmed that SOCS3 protein requires aMTD in order to exhibit anti-cancer efficacy, and the apoptosis-inducing effect of the iCP-SOCS3 recombinant protein is cancer cell-specific. In contrast, the reference drug, 5-FU® hardly induced apoptosis.

Example 21. Investigation of Anti-Cancer Efficacy (Induction of Apoptosis) of iCP-SOCS3

To further determine the effect of iCP-SOCS3 on the tumorigenicity of hepatocellular carcinoma cells, we subsequently investigated whether iCP-SOCS3 regulates apoptosis in Chang liver cells and Huh7 cells.

Apoptotic cells were analyzed using terminal dUTP nick-end labeling (TUNEL) assay with In Situ Cell Death Detection kit TMR red (Roche, 4056 Basel, Switzerland). Cells were treated for 24 hr with 10 μM HS3B or HM₁₆₅S3B proteins with 2% fetal bovine serum and apoptotic cells were visualized by TUNEL staining. Treated cells were washed with cold PBS two times, fixed in 4% paraformaldehyde (PFA, Junsei, Tokyo, Japan) for 1 hr at room temperature, and incubated in 0.1% Triton X-100 for 2 min on the ice. Cells were washed with cold PBS twice, and treated TUNEL reaction mixture for 1 hr at 37° C. in dark, washed cold PBS three times and observed by fluorescence microscopy (Nikon, Tokyo, Japan). Cisplatin® (IC₅₀: 30 μM) was used for reference drug.

The iCP-SOCS3 (HM₁₆₅S3B) protein was considerably efficient inducer of apoptosis in and Huh7 cells (FIG. 70), as assessed by a fluorescent terminal dUTP nick-end labeling (TUNEL) assay. In contrast, iCP-SOCS3 (HM₁₆₅S3B) protein did not induce apoptosis in Chang liver cells (FIG. 69). Consistently, no changes in TUNEL was observed in Chang liver and Huh7 cells treated with HS3B compared to untreated cell (Vehicle). Further, the reference drug, Cisplatin® hardly induced apoptosis.

Example 22. Investigation of Anti-Cancer Efficacy (Inhibition of Cell Cycle Progression) of iCP-SOCS3

To further determine the effect of iCP-SOCS3 on the tumorigenicity of hepatocellular carcinoma cells, we subsequently investigated whether iCP-SOCS3 regulates cell cycle progression in HCCs (Hep3B, HepG2, Huh7 and SK-HEP-1).

HCCs and normal cells were treated with 10 uM protein (Non-CP-SOCS3 (HS3B) and iCP-SOSC3 (HM₁₆₅S3B)) for 8 hrs. After the treatment, cells were washed twice with cold PBS and re-suspended in 1 ml cold PBS, fixed in cold 70% ethanol, washed with cold PBS twice and re-suspended in PI master mix (PI 10 ug/ml (SIGMA, USA), 50 ug/ml RNase A (Invitrogen, USA) in staining buffer) at a final cell density of 2×10⁵ cell/ml. The cell mixtures were incubated 30 min in the dark at 4° C. Flow cytometric analysis was immediately performed using a guava easyCyte™ 8 Instrument (Merck Millipore, Darmstadt, Germany). Cisplatin® (IC50: 30 uM) and 5-FU (IC50: 40 μM) were used for reference drugs.

HM₁₆₅S3B protein (iCP-SOCS3) efficiently inhibited cell cycle progression in HCCs (FIG. 72), while HM₁₆₅S3B protein (iCP-SOCS3) did not inhibit cell cycle progression in normal cells (FIG. 71). Further, the reference drug, Cisplatin® and 5-FU® hardly increased the amount of subG1.

Example 23. Investigation of Anti-Cancer Efficacy (Effect on Molecular Mechanism-Mode of Action) of iCP-SOCS3

To further determine the effect of iCP-SOCS3 on the tumorigenicity of hepatocellular carcinoma cells, we subsequently investigated whether iCP-SOCS3 regulates apoptosis in HepG2 and Hep3B cells.

Cells were treated for 24 hr with 10 μM HS3B or HM₁₆₅S3B proteins, lysed in RIPA lysis buffer containing proteinase inhibitor cocktail, incubated for 15 min at 4° C., and centrifuged at 13,000 rpm for 10 min at 4° C. Equal amounts of lysates were separated on 15% SDS-PAGE gels and transferred to a nitrocellulose membrane. The membranes were blocked using 5% skim milk or 5% Albumin in TBST and incubated with the following antibodies: anti-Bcl-2 (Santa Cruz biotechnology), anti-Cleaved Caspase 3 (Cell Signaling Technology), anti-phospho-p65 and anti-p65 (Cell Signaling Technology), then HRP conjugated anti-mouse or anti-rabbit secondary antibody. The expression of each protein was determined by immunoblotting with indicated antibodies. An antibody against β-actin was used as a loading control.

HepG2 and Hep3B cells treated with HM₁₆₅S3B protein (iCP-SOCS3) dramatically reduced the expression of anti-apoptotic protein such as B-cell lymphoma 2 (Bcl-2) and NF-κB (p-p65) and increased the level of cleaved cysteine-aspartic acid protease (caspase-3) (FIG. 73). These results indicate that iCP-SOCS3 induces apoptosis of hepatocellular carcinoma cells and may suppress the cancer progression by this pathway.

Example 24-1. Investigation of Anti-Cancer Efficacy of iCP-SOCS3 in Xenograft Model (Cell-Derived Xenograft (CDX)-Model)

In the following experiments, western blotting was performed in the same manner as in Example 8-1, and RT-PCR and IHC were performed by the following method.

RT-PCR

Tumor tissues were finely minced using a homogenizer according to the manufacturer's protocol of Hybrid-R (geneall, Korea), and then mRNA was isolated therefrom. 1 ug of mRNA thus separated was used to synthesize cDNA using an Accupower RT Premix (Bioneer, Korea). PCR was performed using 2 ul of cDNA and the primers of Table 45. PCR was performed using an Accupower PCR Premix (Bioneer, Korea) for 30 cycles consisting of denaturing (95° C.), annealing (60° C.), and extending (72° C.) for 45 seconds each.

TABLE 45 Genes Forward Sequence Reverse Sequence Cyclin CCGTTTACAAGCTAAGCAGC GTGGTTCCAAGTCAGAATGC E (SEQ ID NO: 839) (SEQ ID NO: 840) Cyclin AGAAGCTGTGCATCTACACCG GTAGGACAGGAAGTTGTTGGG D1 (SEQ ID NO: 841) (SEQ ID NO: 842) PCNA GAGGCCTGCTGGGATATTAG TACGTGCAAATTCACCAGAAG (SEQ ID NO: 843) (SEQ ID NO: 844) Bax CCCTTTTGCTTCAGGGTTTC GCCACTCGGAAAAAGACCTC (SEQ ID NO: 845) (SEQ ID NO: 846) Bid GTCAAGAAGACATCATCCGG GACCACATCAAGCTTTAGCC (SEQ ID NO: 847) (SEQ ID NO: 848) FAK TGGTGAAAGCTGTCATCGAG CTGGGCCAGTTTCATCTTGT (SEQ ID NO: 849) (SEQ ID NO: 850) VEGF CTTCAAGCCATCCTGTGTGC ACGCGAGTCTGTGTTTTTGC (SEQ ID NO: 851) (SEQ ID NO: 852) GOLPH2 TCTTGGGCTTCAACTACTGG GGGTCTTTAACTGGTCTTGC (SEQ ID NO: 853) (SEQ ID NO: 854) GAPDH AAGGGTCATCATCTCTGCCC GTGATGGCATGGACTGTGGT (SEQ ID NO: 855) (SEQ ID NO: 856)

Immunohistochemistry (IHC) Tissue samples were fixed in 4% Paraformaldehyde (Duksan, South Korea) for 3 days, dehydrated, cleared with xylene and embedded in Paraplast. Sections (6 μm thick) of tumor were placed onto poly-L-lysine coated slides. To block endogenous peroxidase activity, sections were incubated for 15 min with 3% H₂O₂ in methanol. After washing three times with PBS, slides were incubated for 30 min with blocking solution (5% fetal bovine serum in PBS). Mouse anti-Bax antibody (sc-7480, Santa Cruz Biotechnology, SantaCruz, Calif., USA) and rabbit anti-VEGF (ab46154, Abcam, Cambridge, UK) were diluted 1:1000 (to protein concentration 0.1 μg/ml) in blocking solution, applied to sections, and incubated at 4° C. for 24 hrs. After washing three times with PBS, sections were incubated with biotinylated mouse and rabbit IgG (Vector Laboratories, Burlingame, Calif., USA) at a 1:1000 dilution for 1 hr at room temperature, then incubated with avidin-biotinylated peroxidase complex using a Vectorstain ABC Kit (Vector Laboratories, Burlingame, Calif., USA) for 30 min at room temperature. After the slides are reacted with oxidized 3, 3-diaminobenzidine as a chromogen, they were counterstained with Harris hematoxylin (Sigma-Aldrich, USA). Permanently mounted slides were observed and photographed using a microscope equipped with a digital imaging system (ECLIPSE Ti, Nikon, Japan).

Anti-tumor activity of iCP-SOCS3 against human cancer xenografts was assessed. Female Balb/c^(nu/nu) mice (5-week-old, Doo-Yeol Biotech Co. Ltd. Korea) were subcutaneously implanted with Hep3B2.1-7 tumor block (1 mm³) into the left back side of the mouse. Tumor-bearing mice were intravenously administered with 600 μg/head of iCP-SOCS3 (HM₁₆₅S3B) or the control proteins (HS3B) for 21 days and observed for 2 weeks following the termination of the treatment. After protein treatment, mice were killed, and six organs (brain, heart, lung, liver, kidney, and spleen) from each were collected and kept in a suitable fixation solution until the next step. Tumor size was monitored by measuring the longest (length) and shortest dimensions (width) once a day with a dial caliper, and tumor volume was calculated as width2×length×0.5.

As shown in FIG. 74, HM₁₆₅S3B protein significantly suppressed the tumor growth (p<0.05) during the treatment and the effect persisted for at least 2 weeks after the treatment was terminated (80% inhibition at day 21; 70% at day 35, respectively). Whereas, the growth of HS3B-treated tumors increased, matching the rates observed in control mice (Vehicle). These results suggest that iCP-SOCS3 inhibits the growth of established tumors as well as the tumor growth of hepatocellular carcinoma cells.

Example 24-2. Investigation of Anti-Cancer Efficacy of iCP-SOCS3 in Xenograft Model (Cell-Derived Xenograft (CDX)-Model)

Experiments were performed in the same manner as in Example 24-1, except that HepG2 was used as a hepatocellular carcinoma cell line, and tumor growth was measured for 3 weeks after termination of protein administration.

As shown in FIG. 77, HM₁₆₅S3B protein significantly suppressed the tumor growth (p<0.05) during the treatment and the effect persisted for at least 3 weeks after the treatment was terminated (67% at day 42). Whereas, the growth of HS3B-treated tumors increased, matching the rates observed in control mice (Vehicle).

mRNA levels were analyzed using the tumor tissues removed at 2 weeks (Hep3B CDX, day 35) or 3 weeks (HepG2 CDX, day 42) after termination of drug administration to analyze changes in expressions of major biomarker genes by iCP-SOCS3.

Expressions of apoptosis (Bax), cell cycle (cyclinD1, cyclinE), proliferation (PCNA), angiogenesis (VEGF), and migration (FAK)-related genes were significantly regulated by treatment of iCP-SOCS3. Further, expression of GOLPH2 which is a biomarker gene of HCC was remarkably reduced by iCP-SOCS3.

Accordingly, it was confirmed that iCP-SOCS3 is able to inhibit development of hepatocellular carcinoma through expressions of various factors regulating cancer activity such as apoptosis, cell cycle, proliferation, migration, angiogenesis, etc., indicating characteristics of iCP-SOCS3 as a mechanism-specific therapeutic agent targeting hepatocellular carcinoma.

Further, tumors removed at 2 weeks (Hep3B CDX, day 35) or 3 weeks (HepG2 CDX, day 42) after termination of drug administration were used to perform Western blotting and IHC, and increase or decrease in the expressions of cancer activity regulating factors by iCP-SOCS3 was observed.

In iCP-SOCS3-treated tumor tissues, expressions of a cell cycle regulator p21 and an apoptosis inducer Bax were increased, and expression of an apoptosis suppressor Bcl-2 was decreased. Further, expression of an angiogenesis inducer VEGF was also remarkably decreased (FIGS. 74 and 75). Accordingly, it was demonstrated that cell cycle, apoptosis, and angiogenesis are also regulated by iCP-SOCS3 in-vivo.

Example 24-3. Investigation of Anti-Cancer Efficacy of iCP-SOCS3 in Xenograft Model (Cell-Derived Xenograft (CDX)-Model)

In order to examine the effect of iCP-SOCS3 depending on its administration dose, IV injection of iCP-SOCS3 at a dose of 25 mg/kg and 50 mg/kg was performed every day for 3 weeks, followed by observation for 3 weeks. Then, tumor volumes were examined. Further, in order to examine the effect of iCP-SOCS3 depending on its administration frequency, IV injection of iCP-SOCS3 at a dose of 50 mg/kg was performed every day a week and three times a week for 3 weeks, followed by observation for 3 weeks. Then, tumor volumes were examined. Other experimental methods are the same as in Example 24-1.

As shown in FIG. 76, the groups administered with 25 mg/kg and 50 mg/kg of iCP-SOCS3 exhibited 60% and 76% inhibition on the tumor size of hepatocellular carcinoma, respectively, indicating superior anti-cancer efficacy on hepatocellular carcinoma. Further, in order to examine the effect of iCP-SOCS3 depending on its administration frequency, IV injection of iCP-SOCS3 at a dose of 50 mg/kg was performed every day and three times a week for 3 weeks, and as a result, as shown in FIG. 76, the groups administered with iCP-SOCS3 every day and three times a week exhibited 76% and 66% inhibition on the tumor size of hepatocellular carcinoma, respectively.

Example 24-4. Investigation of Anti-Cancer Efficacy of iCP-SOCS3 in Xenograft Model (Cell-Derived Xenograft (CDX)-Model)

IV injection of iCP-SOCS3 at a dose of 50 mg/kg was performed every day for 3 weeks, followed by observation for 3 weeks. Then, tumor volumes were examined. To compare its effect with that of the known therapeutic agent, IP injection of Cisplatin®, which is used for the standard regimen of hepatocellular carcinoma, at a dose of 4 mg/kg was performed twice a week for 3 weeks, followed by observation for 3 weeks. Then, tumor volumes were examined. Other experimental methods are the same as in Example 24-1.

As shown in FIG. 77, iCP-SOCS3 and cisplatin exhibited similar reduction in the tumor volume of hepatocellular carcinoma. However, the cytotoxic agent, cisplatin caused a remarkable weight loss, unlike iCP-SOCS3. Therefore, it was confirmed that iCP-SOCS3 can be developed as a mechanism-specific anti-cancer agent having superior anti-cancer efficacy without adverse effects.

Statistical Analysis

Statistical analysis and graphic presentation have been performed using GraphPad Prism 5.01 software (GraphPad, La Jolla, Calif., USA). All experimental data are presented as means±SEM. Statistical significance was analyzed by the Student's t-test or ANOVA method. Experimental differences between groups were assessed using paired Student's t-tests. For animal experiments, ANOVA was used for comparing between and within groups to determine the significance. Differences with p<0.05 are considered to be statistically significant.

Those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in different forms without departing from the essential characteristics thereof. Therefore, it should be understood that the disclosed embodiments are not limitative, but illustrative in all aspects. The scope of the present invention is made to the appended claims rather than to the foregoing description, and all variations which come within the range of equivalency of the claims are therefore intended to be embraced therein. 

1. An improved Cell-Permeable (iCP)-SOCS3 recombinant protein, which comprises a SOCS3 protein and at least one advanced macromolecule transduction domain (aMTD)(s) being composed of 9˜13 amino acid sequences and having improved cell and/or tissue permeability, wherein the aMTD is fused to one end or both ends of the SOCS3 protein and has the following features of: (a) being composed of 3 or more amino acids sequences selected from the group consisting of Ala, Val, Ile, Leu, and Pro; (b) having proline as amino acid sequences corresponding to any one or more of positions 5 to 8, and 12 of its amino acid sequence; and (c) having an instability index of 40-60; an aliphatic index of 180-220; and a grand average of hydropathy (GRAVY) of 2.1-2.6, as measured by Protparam.
 2. The iCP-SOCS3 recombinant protein according to claim 1, wherein the iCP-SOCS3 recombinant protein further comprises one or more solubilization domain (SD)(s), and the aMTD(s), SOCS3 protein and SD(s) are randomly fused to one another.
 3. The iCP-SOCS3 recombinant protein according to claim 1, wherein the aMTD is composed of 12 amino acid sequences and represented by the following general formula:

Here, X(s) independently refer to Alanine (A), Valine (V), Leucine (L) or Isoleucine (I); and Proline (P) can be positioned in one of U(s) (either 5′, 6′, 7′ or 8′); and the remaining U(s) are independently composed of A, V, L or I, P at the 12′ is Proline.
 4. The iCP-SOCS3 recombinant protein according to claim 2, wherein the iCP-SOCS3 recombinant protein is represented by any one of the following structural formulae: A-B-C, A-C-B, B-A-C, B-C-A, C-A-B, C-B-A, and A-C-B-C wherein A is an advanced macromolecule transduction domain (aMTD) having improved cell and/or tissue permeability, B is a SOCS3 protein, and C is a solubilization domain (SD), and if the iCP-SOCS3 recombinant protein comprises two SDs, the two SDs can be same or different.
 5. The iCP-SOCS3 recombinant protein according to claim 1, wherein the SOCS3 protein has an amino acid sequence of SEQ ID NO:
 814. 6. The iCP-SOCS3 recombinant protein according to claim 5, wherein the SOCS3 protein is encoded by a polynucleotide sequence of SEQ ID NO:
 815. 7. The iCP-SOCS3 recombinant protein according to claim 1, wherein the at least one aMTD(s) has an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 1˜240 and
 822. 8. The iCP-SOCS3 recombinant protein according to claim 7, wherein the at least one aMTD(s) is encoded by a polynucleotide sequence independently selected from the group consisting of SEQ ID NOs: 241˜480 and
 823. 9. The iCP-SOCS3 recombinant protein according to claim 2, wherein the one or more SD(s) has an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 798, 799, 800, 801, 802, 803, and
 804. 10. The iCP-SOCS3 recombinant protein of claim 9, wherein the one or more SD(s) is encoded by a polynucleotide sequence independently selected from the group consisting of SEQ ID NOs: 805, 806, 807, 808, 809, 810, and
 811. 11. The iCP-SOCS3 recombinant protein according to claim 1, wherein the iCP-SOCS3 recombinant protein has a histidine-tag affinity domain additionally fused to one end thereof.
 12. The iCP-SOCS3 recombinant protein according to claim 11, wherein the histidine-tag affinity domain has an amino acid sequence of SEQ ID NO:
 812. 13. The iCP-SOCS3 recombinant protein of claim 12, wherein the histidine-tag affinity domain is encoded by a polynucleotide sequence of SEQ ID NO:
 813. 14. The iCP-SOCS3 recombinant protein according to claim 1, wherein the fusion is formed via a peptide bond or a chemical bond.
 15. The iCP-SOCS3 recombinant protein according to claim 14, wherein the iCP-SOCS3 recombinant protein is used for the treating, preventing, or delaying the onset of, hepatocellular carcinoma.
 16. A polynucleotide sequence encoding the iCP-SOCS3 recombinant protein of claim
 1. 17. A recombinant expression vector comprising the polynucleotide sequence of claim
 16. 18. A transformant transformed with the recombinant expression vector of claim
 17. 19. A preparing method of the iCP-SOCS3 recombinant protein of claim 1 comprising: preparing a recombinant expression vector comprising a polynucleotide sequence encoding the iCP-SOCS3 recombinant protein; preparing a transformant using the recombinant expression vector; culturing the transformant; and recovering the recombinant protein expressed by the culturing.
 20. A method of treating, preventing, or delaying the onset of, hepatocellular carcinoma in a subject comprising: identifying a subject in need of treating, preventing, or delaying the onset of, hepatocellular carcinoma; and administering to the subject a therapeutically effective amount of the iCP-SOCS3 recombinant protein of claim
 1. 21. The iCP-SOCS3 recombinant protein according to claim 5, wherein the one or more SD(s) has an amino acid sequence independently selected from the group consisting of SEQ ID NOs: 798, 799, 800, 801, 802, 803, and
 804. 22. The iCP-SOCS3 recombinant protein of claim 9, wherein the one or more SD(s) is encoded by a polynucleotide sequence independently selected from the group consisting of SEQ ID NOs: 805, 806, 807, 808, 809, 810, and
 811. 